Circular RNA compositions and methods

ABSTRACT

Disclosed herein are circular RNA s and transfer vehicles, along with related compositions and methods of treatment. The circular RNAs can comprise group I intron fragments, spacers, an IRES, duplex forming regions, and/or an expression sequence, thereby having the features of improved expression, functional stability, low immunogenicity, ease of manufacturing, and/or extended half-life compared to linear RNA. Pharmaceutical compositions comprising such circular RNAs and transfer vehicles are particularly suitable for efficient protein expression in immune cells in vivo. Also disclosed are precursor RNAs and materials useful in producing the precursor or circular RNAs, which have improved circularization efficiency and/or are compatible with effective circular RNA purification methods.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.17/503,208, filed on Oct. 15, 2021, which is a continuation of U.S.application Ser. No. 17/202,223, filed on Mar. 15, 2021, which is acontinuation of International Application No. PCT/US2020/063494, filedon Dec. 4, 2020, which claims the benefit of U.S. ProvisionalApplication No. 62/943,779, filed on Dec. 4, 2019; U.S. ProvisionalApplication No. 62/972,194, filed on Feb. 10, 2020; U.S. ProvisionalApplication No. 63/022,248, filed on May 8, 2020; U.S. ProvisionalApplication No. 63/087,582, filed on Oct. 5, 2020; and U.S. ProvisionalApplication No. 62/943,797, filed on Dec. 4, 2019, the contents of eachof which are hereby incorporated by reference in their entirety for allpurposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 2, 2021, isnamed OBS-011WOC3_Sequence_Listing.txt and is 589,830 bytes in size.

BACKGROUND

Conventional gene therapy involves the use of DNA for insertion ofdesired genetic information into host cells. The DNA introduced into thecell is usually integrated to a certain extent into the genome of one ormore transfected cells, allowing for long-lasting action of theintroduced genetic material in the host. While there may be substantialbenefits to such sustained action, integration of exogenous DNA into ahost genome may also have many deleterious effects. For example, it ispossible that the introduced DNA will be inserted into an intact gene,resulting in a mutation which impedes or even totally eliminates thefunction of the endogenous gene. Thus, gene therapy with DNA may resultin the impairment of a vital genetic function in the treated host, suchas, e.g., elimination or deleteriously reduced production of anessential enzyme or interruption of a gene critical for the regulationof cell growth, resulting in unregulated or cancerous cellproliferation. In addition, with conventional DNA based gene therapy itis necessary for effective expression of the desired gene product toinclude a strong promoter sequence, which again may lead to undesirablechanges in the regulation of normal gene expression in the cell. It isalso possible that the DNA based genetic material will result in theinduction of undesired anti-DNA antibodies, which in turn, may trigger apossibly fatal immune response. Gene therapy approaches using viralvectors can also result in an adverse immune response. In somecircumstances, the viral vector may even integrate into the host genome.In addition, production of clinical grade viral vectors is alsoexpensive and time consuming. Targeting delivery of the introducedgenetic material using viral vectors can also be difficult to control.Thus, while DNA based gene therapy has been evaluated for delivery ofsecreted proteins using viral vectors (U.S. Pat. No. 6,066,626; US2004/0110709), these approaches may be limited for these variousreasons.

In contrast to DNA, the use of RNA as a gene therapy agent issubstantially safer because RNA does not involve the risk of beingstably integrated into the genome of the transfected cell, thuseliminating the concern that the introduced genetic material willdisrupt the normal functioning of an essential gene, or cause a mutationthat results in deleterious or oncogenic effects, and extraneouspromoter sequences are not required for effective translation of theencoded protein, again avoiding possible deleterious side effects. Inaddition, it is not necessary for mRNA to enter the nucleus to performits function, while DNA must overcome this major barrier.

Circular RNA is useful in the design and production of stable forms ofRNA. The circularization of an RNA molecule provides an advantage to thestudy of RNA structure and function, especially in the case of moleculesthat are prone to folding in an inactive conformation (Wang and Ruffner,1998). Circular RNA can also be particularly interesting and useful forin vivo applications, especially in the research area of RNA-basedcontrol of gene expression and therapeutics, including proteinreplacement therapy and vaccination.

Prior to this invention, there were three main techniques for makingcircularized RNA in vitro: the splint-mediated method, the permutedintron-exon method, and the RNA ligase-mediated method. However, theexisting methodologies are limited by the size of RNA that can becircularized, thus limiting their therapeutic application.

SUMMARY

The present application provides circular RNAs and transfer vehicles,along with related compositions and methods of treatment. The transfervehicles can comprise, e.g., ionizable lipid, PEG-modified lipid, and/orstructural lipid, thereby forming lipid nanoparticles encapsulating thecircular RNAs. The circular RNAs can comprise group I intron fragments,spacers, an IRES, duplex forming regions, and/or an expression sequence,thereby having the features of improved expression, functionalstability, low immunogenicity, ease of manufacturing, and/or extendedhalf-life compared to linear RNA. Pharmaceutical compositions comprisingsuch circular RNAs and transfer vehicles are particularly suitable forefficient protein expression in immune cells in vivo. The presentapplication also provides precursor RNAs and materials useful inproducing the precursor or circular RNAs, which have improvedcircularization efficiency and/or are compatible with effective circularRNA purification methods.

Accordingly, one aspect of the present application provides apharmaceutical composition comprising a circular RNA polynucleotide anda transfer vehicle comprising an ionizable lipid represented by Formula(1):

wherein:

-   -   each n is independently an integer from 2-15;    -   L₁ and L₃ are each independently —OC(O)—* or —C(O)O—*, wherein        “*” indicates the attachment point to R₁ or R₃;    -   R₁ and R₃ are each independently a linear or branched C₉-C₂₀        alkyl or C₉-C₂₀ alkenyl, optionally substituted by one or more        substituents selected from a group consisting of oxo, halo,        hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl,        hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl,        aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl,        (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl,        alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino,        aminoalkylcarbonylamino, aminocarbonylalkylamino,        (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino,        hydroxycarbonyl, alkyloxycarbonyl, aminocarbonyl,        aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl,        dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl,        (alkylaminoalkyl)(alkyl)amino carbonyl, alkylaminoalkylcarbonyl,        dialkylaminoalkylcarbonyl, heterocyclylcarbonyl,        alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide,        alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; and    -   R₂ is selected from a group consisting of:

In some embodiments, R₁ and R₃ are each independently selected from agroup consisting of

In some embodiments, R₁ and R₃ are the same. In some embodiments, R₁ andR₃ are different.

In some embodiments, the ionizable lipid of Formula (1) is representedby Formula (1-1) or Formula (1-2):

In some embodiments, the ionizable lipid is selected from the groupconsisting of:

In another aspect, the present application provides a pharmaceuticalcomposition comprising: a circular RNA polynucleotide and a transfervehicle comprising an ionizable lipid represented by Formula (2):

wherein:

-   -   each n is independently an integer from 1-15;    -   R₁ and R₂ are each independently selected from a group        consisting of:

-   -    and    -   R₃ is selected from a group consisting of:

In another aspect, the present application provides a pharmaceuticalcomposition comprising: a circular RNA polynucleotide, and a transfervehicle comprising an ionizable lipid represented by Formula (3):

wherein:

-   -   X is selected from —O—, —S—, or —OC(O)—*, wherein * indicates        the attachment point to R₁;    -   R₁ is selected from a group consisting of:

-   -    and    -   R₂ is selected from a group consisting of:

In some embodiments, the ionizable lipid of Formula (3) is representedby Formula (3-1), Formula (3-2), or Formula (3-3):

In another aspect, the present application provides a pharmaceuticalcomposition comprising: a circular RNA polynucleotide, and a transfervehicle comprising an ionizable lipid represented by Formula (4):

wherein: each n is independently an integer from 2-15; and R₂ is definedin Formula (1).

In another aspect, the present application provides a pharmaceuticalcomposition comprising: a circular RNA polynucleotide, and a transfervehicle comprising an ionizable lipid selected from Table 10a.

In some embodiments, the circular RNA comprises a first expressionsequence. In some embodiments, the first expression sequence encodes atherapeutic protein. In some embodiments, the first expression sequenceencodes a cytokine or a functional fragment thereof. In someembodiments, the first expression sequence encodes a transcriptionfactor. In some embodiments, the first expression sequence encodes animmune checkpoint inhibitor. In some embodiments, the first expressionsequence encodes a chimeric antigen receptor.

In some embodiments, the circular RNA polynucleotide further comprises asecond expression sequence. In some embodiments, the circular RNApolynucleotide further comprises an internal ribosome entry site (IRES).

In some embodiments, the first and second expression sequences areseparated by a ribosomal skipping element or a nucleotide sequenceencoding a protease cleavage site. In some embodiments, the firstexpression sequence encodes a first T-cell receptor (TCR) chain and thesecond expression sequence encodes a second TCR chain.

In some embodiments, the circular RNA polynucleotide comprises one ormore microRNA binding sites. the microRNA binding site is recognized bya microRNA expressed in the liver. In some embodiments, the microRNAbinding site is recognized by miR-122.

In some embodiments, the circular RNA polynucleotide comprises a firstIRES associated with greater protein expression in a human immune cellthan in a reference human cell. In some embodiments, the human immunecell is a T cell, an NK cell, an NKT cell, a macrophage, or aneutrophil. In some embodiments, the reference human cell is a hepaticcell.

In some embodiments, the circular RNA polynucleotide comprises, in thefollowing order: a) a post-splicing intron fragment of a 3′ group Iintron fragment, b) an IRES, c) an expression sequence, and d) apost-splicing intron fragment of a 5′ group I intron fragment. In someembodiments, the circular RNA polynucleotide comprises. In someembodiments, the circular RNA polynucleotide comprises a first spacerbefore the post-splicing intron fragment of the 3′ group I intronfragment, and a second spacer after the post-splicing intron fragment ofthe 5′ group I intron fragment. In some embodiments, the first andsecond spacers each have a length of about 10 to about 60 nucleotides.

In some embodiments, the circular RNA polynucleotide is made viacircularization of a RNA polynucleotide comprising, in the followingorder: a 3′ group I intron fragment, an IRES, an expression sequence,and a 5′ group I intron fragment.

In some embodiments, the circular RNA polynucleotide is made viacircularization of a RNA polynucleotide comprising, in the followingorder: a 5′ external duplex forming region, a 3′ group I intronfragment, a 5′ internal spacer optionally comprising a 5′ internalduplex forming region, an IRES, an expression sequence, a 3′ internalspacer optionally comprising a 3′ internal duplex forming region, a 5′group I intron fragment, and a 3′ external duplex forming region.

In some embodiments, the circular RNA polynucleotide is made viacircularization of a RNA polynucleotide comprising, in the followingorder: a 5′ external duplex forming region, a 5′ external spacer, a 3′group I intron fragment, a 5′ internal spacer optionally comprising a 5′internal duplex forming region, an IRES, an expression sequence, a 3′internal spacer optionally comprising a 3′ internal duplex formingregion, a 5′ group I intron fragment, a 3′ external spacer, and a 3′external duplex forming region.

In some embodiments, the circular RNA polynucleotide is made viacircularization of a RNA polynucleotide comprising, in the followingorder: a 3′ group I intron fragment, a 5′ internal spacer comprising a5′ internal duplex forming region, an IRES, an expression sequence, a 3′internal spacer comprising a 3′ internal duplex forming region, and a 5′group I intron fragment.

In some embodiments, the circular RNA polynucleotide is made viacircularization of a RNA polynucleotide comprising, in the followingorder: a 5′ external duplex forming region, a 5′ external spacer, a 3′group I intron fragment, a 5′ internal spacer comprising a 5′ internalduplex forming region, an IRES, an expression sequence, a 3′ internalspacer comprising a 3′ internal duplex forming region, a 5′ group Iintron fragment, a 3′ external spacer, and a 3′ external duplex formingregion.

In some embodiments, the circular RNA polynucleotide is made viacircularization of a RNA polynucleotide comprising, in the followingorder: a first polyA sequence, a 5′ external duplex forming region, a 5′external spacer, a 3′ group I intron fragment, a 5′ internal spacercomprising a 5′ internal duplex forming region, an IRES, an expressionsequence, a 3′ internal spacer comprising a 3′ internal duplex formingregion, a 5′ group I intron fragment, a 3′ external spacer, a 3′external duplex forming region, and a second polyA sequence.

In some embodiments, the circular RNA polynucleotide is made viacircularization of a RNA polynucleotide comprising, in the followingorder: a first polyA sequence, a 5′ external spacer, a 3′ group I intronfragment, a 5′ internal spacer comprising a 5′ internal duplex formingregion, an IRES, an expression sequence, a 3′ internal spacer comprisinga 3′ internal duplex forming region, a 5′ group I intron fragment, a 3′external spacer, and a second polyA sequence.

In some embodiments, the circular RNA polynucleotide is made viacircularization of a RNA polynucleotide comprising, in the followingorder: a first polyA sequence, a 5′ external spacer, a 3′ group I intronfragment, a 5′ internal spacer comprising a 5′ internal duplex formingregion, an IRES, an expression sequence, a stop condon, a 3′ internalspacer comprising a 3′ internal duplex forming region, a 5′ group Iintron fragment, a 3′ external spacer, and a second polyA sequence.

In some embodiments, at least one of the 3′ or 5′ internal or externalspacers has a length of about 8 to about 60 nucleotides. In someembodiments, the 3′ and 5′ external duplex forming regions each has alength of about 10-50 nucleotides. In some embodiments, the 3′ and 5′internal duplex forming regions each has a length of about 6-30nucleotides.

In some embodiments, the IRES is selected from Table 17, or is afunctional fragment or variant thereof. In some embodiments, the IREShas a sequence of an IRES from Taura syndrome virus, Triatoma virus,Theiler's encephalomyelitis virus, Simian Virus 40, Solenopsis invictavirus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, Humanpoliovirus 1, Plautia stali intestine virus, Kashmir bee virus, Humanrhinovirus 2, Homalodisca coagulata virus-1, Human ImmunodeficiencyVirus type 1, Homalodisca coagulata virus-1, Himetobi P virus, HepatitisC virus, Hepatitis A virus, Hepatitis GB virus, Foot and mouth diseasevirus, Human enterovirus 71, Equine rhinitis virus, Ectropis obliquapicorna-like virus, Encephalomyocarditis virus, Drosophila C Virus,Human coxsackievirus B3, Crucifer tobamovirus, Cricket paralysis virus,Bovine viral diarrhea virus 1, Black Queen Cell Virus, Aphid lethalparalysis virus, Avian encephalomyelitis virus, Acute bee paralysisvirus, Hibiscus chlorotic ringspot virus, Classical swine fever virus,Human FGF2, Human SFTPA1, Human AML1/RUNX1, Drosophila antennapedia,Human AQP4, Human AT1R, Human BAG-1, Human BCL2, Human BiP, Humanc-IAP1, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIF1alpha, Human n.myc, Mouse Gtx, Human p27kip1, Human PDGF2/c-sis, Humanp53, Human Pim-1, Mouse Rbm3, Drosophila reaper, Canine Scamper,Drosophila Ubx, Human UNR, Mouse UtrA, Human VEGF-A, Human XIAP,Drosophila hairless, S. cerevisiae TFIID, S. cerevisiae YAP1, tobaccoetch virus, turnip crinkle virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCVpEC9, Picobirnavirus, HCV QC64, Human Cosavirus E/D, Human Cosavirus F,Human Cosavirus JMY, Rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21,Salivirus A SH1, Salivirus FHB, Salivirus NG-J1, Human Parechovirus 1,Crohivirus B, Yc-3, Rosavirus M-7, Shanbavirus A, Pasivirus A, PasivirusA 2, Echovirus E14, Human Parechovirus 5, Aichi Virus, Hepatitis A VirusHA16, Phopivirus, CVA10, Enterovirus C, Enterovirus D, Enterovirus J,Human Pegivirus 2, GBV-C GT110, GBV-C K1737, GBV-C Iowa, Pegivirus A1220, Pasivirus A 3, Sapelovirus, Rosavirus B, Bakunsa Virus, TremovirusA, Swine Pasivirus 1, PLV-CHN, Pasivirus A, Sicinivirus, Hepacivirus K,Hepacivirus A, BVDV1, Border Disease Virus, BVDV2, CSFV-PK15C, SF573Dicistrovirus, Hubei Picorna-like Virus, CRPV, Apodemus AgrariusPicornavirus, Caprine Kobuvirus, Parabovirus, Salivirus A BN5, SalivirusA BN2, Salivirus A 02394, Salivirus A GUT, Salivirus A CH, Salivirus ASZ1, Salivirus FHB, CVB3, CVB1, Echovirus 7, CVB5, EVA71, CVA3, CVA12,EV24, or an aptamer to eIF4G.

In some embodiments, the first and second polyA sequences each have alength of about 15-50 nt. In some embodiments, the first and secondpolyA sequences each have a length of about 20-25 nt.

In some embodiments, the circular RNA polynucleotide contains at leastabout 80%, at least about 90%, at least about 95%, or at least about 99%naturally occurring nucleotides. In some embodiments, the circular RNApolynucleotide consists of naturally occurring nucleotides.

In some embodiments, the expression sequence is codon optimized. In someembodiments, the circular RNA polynucleotide is optimized to lack atleast one microRNA binding site present in an equivalent pre-optimizedpolynucleotide. In some embodiments, the circular RNA polynucleotide isoptimized to lack at least one microRNA binding site capable of bindingto a microRNA present in a cell within which the circular RNApolynucleotide is expressed. In some embodiments, the circular RNApolynucleotide is optimized to lack at least one endonucleasesusceptible site present in an equivalent pre-optimized polynucleotide.In some embodiments, the circular RNA polynucleotide is optimized tolack at least one endonuclease susceptible site capable of being cleavedby an endonuclease present in a cell within which the endonuclease isexpressed. In some embodiments, the circular RNA polynucleotide isoptimized to lack at least one RNA editing susceptible site present inan equivalent pre-optimized polynucleotide.

In some embodiments, the circular RNA polynucleotide is from about 100nt to about 10,000 nt in length. In some embodiments, the circular RNApolynucleotide is from about 100 nt to about 15,000 nt in length. Insome embodiments, the circular RNA is more compact than a referencelinear RNA polynucleotide having the same expression sequence as thecircular RNA polynucleotide.

In some embodiments, the pharmaceutical composition has a duration oftherapeutic effect in a human cell greater than or equal to that of acomposition comprising a reference linear RNA polynucleotide having thesame expression sequence as the circular RNA polynucleotide. In someembodiments, the reference linear RNA polynucleotide is a linear,unmodified or nucleoside-modified, fully-processed mRNA comprising acap1 structure and a polyA tail at least 80 nt in length.

In some embodiments, the pharmaceutical composition has a duration oftherapeutic effect in vivo in humans greater than that of a compositioncomprising a reference linear RNA polynucleotide having the sameexpression sequence as the circular RNA polynucleotide. In someembodiments, the pharmaceutical composition has an duration oftherapeutic effect in vivo in humans of at least about 10, at leastabout 20, at least about 30, at least about 40, at least about 50, atleast about 60, at least about 70, at least about 80, at least about 90,or at least about 100 hours.

In some embodiments, the pharmaceutical composition has a functionalhalf-life in a human cell greater than or equal to that of apre-determined threshold value. In some embodiments, the pharmaceuticalcomposition has a functional half-life in vivo in humans greater thanthat of a pre-determined threshold value. In some embodiments, thefunctional half-life is determined by a functional protein assay. Insome embodiments, the functional protein assay is an in vitro luciferaseassay. In some embodiments, the functional protein assay comprisesmeasuring levels of protein encoded by the expression sequence of thecircular RNA polynucleotide in a patient serum or tissue sample. In someembodiments, wherein the pre-determined threshold value is thefunctional half-life of a reference linear RNA polynucleotide comprisingthe same expression sequence as the circular RNA polynucleotide. In someembodiments, the pharmaceutical composition has a functional half-lifeof at least about 20 hours.

In some embodiments, the pharmaceutic composition comprises a structurallipid and a PEG-modified lipid. In some embodiments, the structurallipid binds to C1q and/or promotes the binding of the transfer vehiclecomprising said lipid to C1q compared to a control transfer vehiclelacking the structural lipid and/or increases uptake of C1q-boundtransfer vehicle into an immune cell compared to a control transfervehicle lacking the structural lipid. In some embodiments, the immunecell is a T cell, an NK cell, an NKT cell, a macrophage, or aneutrophil.

In some embodiments, the structural lipid is cholesterol. In someembodiments, the structural lipid is beta-sitosterol. In someembodiments, the structural lipid is not beta-sitosterol.

In some embodiments, the PEG-modified lipid is DSPE-PEG, DMG-PEG, orPEG-1. In some embodiments, the PEG-modified lipid is DSPE-PEG(2000).

In some embodiments, the pharmaceutic composition further comprises ahelper lipid. In some embodiments, the helper lipid is DSPC or DOPE.

In some embodiments, the pharmaceutic composition comprises DOPE,cholesterol, and DSPE-PEG.

In some embodiments, the transfer vehicle comprises about 0.5% to about4% PEG-modified lipids by molar ratio. In some embodiments, the transfervehicle comprises about 1% to about 2% PEG-modified lipids by molarratio.

In some embodiments, the transfer vehicle comprises

-   -   a. an ionizable lipid is represented by

-   -   b. DOPE,    -   c. cholesterol, and    -   d. DSPE-PEG(2000).

In some embodiments, the molar ration of ionizablelipid:DSPC:cholesterol:DSPE-PEG(2000) is 62:4:33:1.

In some embodiments, the transfer vehicle comprises

-   -   a. an ionizable lipid is represented by

-   -   b. DOPE,    -   c. cholesterol, and    -   d. DSPE-PEG(2000).

In some embodiments, the molar ration of ionizablelipid:DSPC:cholesterol:DSPE-PEG(2000) is 50:10:38.5:1.5.

In some embodiments, the transfer vehicle has a nitrogen:phosphate (N:P)ratio of about 3 to about 6.

In some embodiments, the transfer vehicle is formulated for endosomalrelease of the circular RNA polynucleotide.

In some embodiments, the transfer vehicle is capable of binding to APOE.In some embodiments, the transfer vehicle interacts with apolipoproteinE (APOE) less than an equivalent transfer vehicle loaded with areference linear RNA having the same expression sequence as the circularRNA polynucleotide. In some embodiments, the exterior surface of thetransfer vehicle is substantially free of APOE binding sites.

In some embodiments, the transfer vehicle has a diameter of less thanabout 120 nm. In some embodiments, the transfer vehicle does not formaggregates with a diameter of more than 300 nm.

In some embodiments, the transfer vehicle has an in vivo half-life ofless than about 30 hours.

In some embodiments, the transfer vehicle is capable of low densitylipoprotein receptor (LDLR) dependent uptake into a cell. In someembodiments, the transfer vehicle is capable of LDLR independent uptakeinto a cell.

In some embodiments, the pharmaceutical composition is substantiallyfree of linear RNA.

In some embodiments, the pharmaceutical composition further comprises atargeting moiety operably connected to the transfer vehicle. In someembodiments, the targeting moiety specifically binds an immune cellantigen or indirectly. In some embodiments, the immune cell antigen is aT cell antigen. In some embodiments, the T cell antigen is selected fromthe group consisting of CD2, CD3, CD5, CD7, CD8, CD4, beta7 integrin,beta2 integrin, and C1q.

In some embodiments, the pharmaceutical composition further comprises anadapter molecule comprising a transfer vehicle binding moiety and a cellbinding moiety, wherein the targeting moiety specifically binds thetransfer vehicle binding moiety and the cell binding moiety specificallybinds a target cell antigen. In some embodiments, the target cellantigen is an immune cell antigen. In some embodiments, the immune cellantigen is a T cell antigen, an NK cell, an NKT cell, a macrophage, or aneutrophil. In some embodiments, the T cell antigen is selected from thegroup consisting of CD2, CD3, CD5, CD7, CD8, CD4, beta7 integrin, beta2integrin, CD25, CD39, CD73, A2a Receptor, A2b Receptor, and C1q. In someembodiments, the immune cell antigen is a macrophage antigen. In someembodiments, the macrophage antigen is selected from the groupconsisting of mannose receptor, CD206, and C1q.

In some embodiments, the targeting moiety is a small molecule. In someembodiments, the small molecule binds to an ectoenzyme on an immunecell, wherein the ectoenzyme is selected from the group consisting ofCD38, CD73, adenosine 2a receptor, and adenosine 2b receptor. In someembodiments, the small molecule is mannose, a lectin, acivicin, biotin,or digoxigenin.

In some embodiments, the targeting moiety is a single chain Fv (scFv)fragment, nanobody, peptide, peptide-based macrocycle, minibody, smallmolecule ligand such as folate, arginylglycylaspartic acid (RGD), orphenol-soluble modulin alpha 1 peptide (PSMA1), heavy chain variableregion, light chain variable region or fragment thereof.

In some embodiments, the ionizable lipid has a half-life in a cellmembrane less than about 2 weeks. In some embodiments, the ionizablelipid has a half-life in a cell membrane less than about 1 week. In someembodiments, the ionizable lipid has a half-life in a cell membrane lessthan about 30 hours. In some embodiments, the ionizable lipid has ahalf-life in a cell membrane less than the functional half-life of thecircular RNA polynucleotide.

In another aspect, the present application provides a method of treatingor preventing a disease, disorder, or condition, comprisingadministering an effective amount of a pharmaceutical compositiondisclosed herein. In some embodiments, the disease, disorder, orcondition is associated with aberrant expression, activity, orlocalization of a polypeptide selected from Tables 27 or 28. In someembodiments, the circular RNA polynucleotide encodes a therapeuticprotein. In some embodiments, therapeutic protein expression in thespleen is higher than therapeutic protein expression in the liver. Insome embodiments, therapeutic protein expression in the spleen is atleast about 2.9× therapeutic protein expression in the liver. In someembodiments, the therapeutic protein is not expressed at functionallevels in the liver. In some embodiments, the therapeutic protein is notexpressed at detectable levels in the liver. In some embodiments,therapeutic protein expression in the spleen is at least about 63% oftotal therapeutic protein expression.

In another aspect, the present application provides a linear RNApolynucleotide comprising, from 5′ to 3′, a 3′ group I intron fragment,an Internal Ribosome Entry Site (IRES), an expression sequence, and a 5′group I intron fragment, further comprising a first spacer 5′ to the 3′group I intron fragment and/or a second spacer 3′ to the 5′ group Iintron fragment.

In some embodiments, the linear RNA polynucleotide comprises a firstspacer 5′ to the 3′ group I intron fragment. In some embodiments, thefirst spacer has a length of 10-50 nucleotides, optionally 10-20nucleotides, further optionally about 15 nucleotides. In someembodiments, the first spacer comprises a polyA sequence.

In some embodiments, the linear RNA polynucleotide comprises a secondspacer 3′ to the 5′ group I intron fragment. In some embodiments, thesecond spacer has a length of 10-50 nucleotides, optionally 10-20nucleotides, further optionally about 15 nucleotides. In someembodiments, the second spacer comprises a polyA sequence.

In some embodiments, the linear RNA polynucleotide further comprises athird spacer between the 3′ group I intron fragment and IRES. In someembodiments, the third spacer has a length of about 10 to about 60nucleotides. In some embodiments, the linear RNA polynucleotide furthercomprises a first and a second duplex forming regions capable of forminga duplex. In some embodiments, the first and second duplex formingregions each have a length of about 9 to 19 nucleotides. In someembodiments, the first and second duplex forming regions each have alength of about 30 nucleotides.

In some embodiments, the linear RNA polynucleotide has enhancedexpression, circularization efficiency, functional stability, and/orstability as compared to a reference linear RNA polynucleotide, whereinthe reference linear RNA polynucleotide comprises, from 5′ to 3′, afirst polyA sequence, a 5′ external spacer, a 3′ group I intronfragment, a 5′ internal spacer comprising a 5′ internal duplex formingregion, an IRES, an expression sequence, a stop condon, a 3′ internalspacer comprising a 3′ internal duplex forming region, a 5′ group Iintron fragment, a 3′ external spacer, and a second polyA sequence.

In some embodiments, the linear RNA polynucleotide has enhancedexpression, circularization efficiency, functional stability, and/orstability as compared to a reference linear RNA polynucleotide, whereinthe reference linear RNA polynucleotide comprises, from 5′ to 3′, areference 3′ group I intron fragment, a reference IRES, a referenceexpression sequence, and a reference 5′ group I intron fragment, anddoes not comprise a spacer 5′ to the 3′ group I intron fragment or aspacer 3′ to the 5′ group I intron fragment. In some embodiments, theexpression sequence and the reference expression sequence have the samesequence. In some embodiments, the IRES and the reference IRES have thesame sequence.

In some embodiments, the linear RNA polynucleotide comprises a 3′Anabaena group I intron fragment and a 5′ Anabaena group I intronfragment. In some embodiments, the reference RNA polynucleotidecomprises a reference 3′ Anabaena group I intron fragment and areference 5′ Anabaena group I intron fragment. In some embodiments, thereference 3′ Anabaena group I intron fragment and reference 5′ Anabaenagroup I intron fragment were generated using the L6-5 permutation site.In some embodiments, the 3′ Anabaena group I intron fragment and 5′Anabaena group I intron fragment were not generated using the L6-5permutation site. In some embodiments, the 3′ Anabaena group I intronfragment comprises or consists of a sequence selected from SEQ ID NO:112-123 and 125-150. In some embodiments, the 5′ Anabaena group I intronfragment comprises a corresponding sequence selected from SEQ ID NO:73-84 and 86-111. In some embodiments, the 5′ Anabaena group I intronfragment comprises or consists of a sequence selected from SEQ ID NO:73-84 and 86-111. In some embodiments, the 3′ Anabaena group I intronfragment comprises or consists of a corresponding sequence selected fromSEQ ID NO: 112-124 and 125-150.

In some embodiments, the IRES comprises a nucleotide sequence selectedfrom SEQ ID NOs: 348-351. In some embodiments, the reference IRES isCVB3. In some embodiments, the IRES is not CVB3. In some embodiments,the IRES comprises a sequence selected from SEQ ID NOs: 1-64 and 66-72.

In another aspect, the present application discloses a circular RNApolynucleotide produced from the linear RNA disclosed herein.

In another aspect, the present application discloses a circular RNAcomprising, from 5′ to 3′, a 3′ group I intron fragment, an IRES, anexpression sequence, and a 5′ group I intron fragment, wherein the IREScomprises a nucleotide sequence selected from SEQ ID NOs: 348-351.

In some embodiments, the circular RNA polynucleotide further comprises aspacer between the 3′ group I intron fragment and the IRES.

In some embodiments, the circular RNA polynucleotide further comprises afirst and a second duplex forming regions capable of forming a duplex.In some embodiments, the first and second duplex forming regions eachhave a length of about 9 to 19 nucleotides. In some embodiments, thefirst and second duplex forming regions each have a length of about 30nucleotides.

In some embodiments, the expression sequence has a size of at leastabout 1,000 nt, at least about 2,000 nt, at least about 3,000 nt, atleast about 4,000 nt, or at least about 5,000 nt.

In some embodiments, the RNA polynucleotide comprises naturalnucleotides. In some embodiments, the expression sequence is codonoptimized. In some embodiments, the RNA polynucleotide further comprisesa translation termination cassette comprising at least one stop codon ineach reading frame. In some embodiments, the translation terminationcassette comprises at least two stop codons in the reading frame of theexpression sequence. In some embodiments, the RNA polynucleotide isoptimized to lack at least one microRNA binding site present in anequivalent pre-optimized polynucleotide. In some embodiments, the RNApolynucleotide is optimized to lack at least one endonucleasesusceptible site present in an equivalent pre-optimized polynucleotide.In some embodiments, the RNA polynucleotide is optimized to lack atleast one RNA editing susceptible site present in an equivalentpre-optimized polynucleotide.

In some embodiments, the RNA polynucleotide comprises at least 2expression sequences. In some embodiments, each expression sequenceencodes a different therapeutic protein.

In some embodiments, a circular RNA polynucleotide disclosed herein isfrom about 100 to 15,000 nucleotides, optionally about 100 to 12,000nucleotides, further optionally about 100 to 10,000 nucleotides inlength.

In some embodiments, a circular RNA polynucleotide disclosed herein hasan in vivo duration of therapeutic effect in humans of at least about 20hours. In some embodiments, a circular RNA polynucleotide disclosedherein has a functional half-life of at least about 20 hours. In someembodiments, the circular RNA polynucleotide has a duration oftherapeutic effect in a human cell greater than or equal to that of anequivalent linear RNA polynucleotide comprising the same expressionsequence. In some embodiments, the circular RNA polynucleotide has afunctional half-life in a human cell greater than or equal to that of anequivalent linear RNA polynucleotide comprising the same expressionsequence. In some embodiments, the circular RNA polynucleotide has an invivo duration of therapeutic effect in humans greater than that of anequivalent linear RNA polynucleotide having the same expressionsequence. In some embodiments, the circular RNA polynucleotide has an invivo functional half-life in humans greater than that of an equivalentlinear RNA polynucleotide having the same expression sequence.

In another aspect, the present disclosure provides a compositioncomprising a circular RNA polynucleotide disclosed herein, ananoparticle, and optionally, a targeting moiety operably connected tothe nanoparticle. In some embodiments, the nanoparticle is a lipidnanoparticle, a core-shell nanoparticle, a biodegradable nanoparticle, abiodegradable lipid nanoparticle, a polymer nanoparticle, or abiodegradable polymer nanoparticle. In some embodiments, thepharmaceutical composition comprises a targeting moiety, wherein thetargeting moiety mediates receptor-mediated endocytosis or direct fusionselectively into cells of a selected cell population or tissue in theabsence of cell isolation or purification. In some embodiments, thetargeting moiety is a scfv, nanobody, peptide, minibody, polynucleotideaptamer, heavy chain variable region, light chain variable region orfragment thereof. In some embodiments, wherein less than 1%, by weight,of the polynucleotides in the composition are double stranded RNA, DNAsplints, or triphosphorylated RNA. In some embodiments, less than 1%, byweight, of the polynucleotides and proteins in the pharmaceuticalcomposition are double stranded RNA, DNA splints, triphosphorylated RNA,phosphatase proteins, protein ligases, and capping enzymes.

In another aspect, the present disclosure provides a method of treatinga subject in need thereof comprising administering a therapeuticallyeffective amount of a composition comprising the circular RNApolynucleotide disclosed herein, a nanoparticle, and optionally, atargeting moiety operably connected to the nanoparticle.

In another aspect, the present disclosure provides a method of treatinga subject in need thereof comprising administering a therapeuticallyeffective amount of the pharmaceutical composition disclosed herein. Insome embodiments, the targeting moiety is an scfv, nanobody, peptide,minibody, heavy chain variable region, light chain variable region, anextracellular domain of a TCR, or a fragment thereof. In someembodiments, the nanoparticle is a lipid nanoparticle, a core-shellnanoparticle, or a biodegradable nanoparticle. In some embodiments, thenanoparticle comprises one or more cationic lipids, ionizable lipids, orpoly β-amino esters. In some embodiments, the nanoparticle comprises oneor more non-cationic lipids. In some embodiments, the nanoparticlecomprises one or more PEG-modified lipids, polyglutamic acid lipids, orHyaluronic acid lipids. In some embodiments, the nanoparticle comprisescholesterol. In some embodiments, the nanoparticle comprises arachidonicacid or oleic acid.

In some embodiments, a provided pharmaceutical composition comprises atargeting moiety, wherein the targeting moiety mediatesreceptor-mediated endocytosis selectively into cells of a selected cellpopulation in the absence of cell selection or purification.

In some embodiments, a provided nanoparticle comprises more than onecircular RNA polynucleotide.

In another aspect, the present application provides a DNA vectorencoding the RNA polynucleotide disclosed herein. In some embodiments,the DNA vector further comprises a transcription regulatory sequence. Insome embodiments, the transcription regulatory sequence comprises apromoter and/or an enhancer. In some embodiments, the promoter comprisesa T7 promoter. In some embodiments, the DNA vector comprises a circularDNA. In some embodiments, the DNA vector comprises a linear DNA.

In another aspect, the present application provides a prokaryotic cellcomprising the DNA vector disclosed herein.

In another aspect, the present application provides a eukaryotic cellcomprising the circular RNA polynucleotide disclosed herein. In someembodiments, the eukaryotic cell is a human cell.

In another aspect, the present application provides a method ofproducing a circular RNA polynucleotide, the method comprisingincubating the linear RNA polynucleotide disclosed herein under suitableconditions for circularization. In some embodiments, the methodcomprises incubating the DNA disclosed herein under suitable conditionsfor transcription. In some embodiments, the DNA is transcribed in vitro.In some embodiments, the suitable conditions comprises adenosinetriphosphate (ATP), guanine triphosphate (GTP), cytosine triphosphate(CTP), uridine triphosphate (UTP), and an RNA polymerase. In someembodiments, the suitable conditions further comprises guaninemonophosphate (GMP). In some embodiments, the ratio of GMP concentrationto GTP concentration is within the range of about 3:1 to about 15:1,optionally about 4:1, 5:1, or 6:1.

In another aspect, the present application provides a method ofproducing a circular RNA polynucleotide, the method comprising culturingthe prokaryotic cell disclosed herein under suitable conditions fortranscribing the DNA in the cell. In some embodiments, the methodfurther comprising purifying a circular RNA polynucleotide. In someembodiments, the circular RNA polynucleotide is purified by negativeselection using an affinity oligonucleotide that hybridizes with thefirst or second spacer conjugated to a solid surface. In someembodiments, the first or second spacer comprises a polyA sequence, andwherein the affinity oligonucleotide is a deoxythymine oligonucleotide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, 1C, 1D, and 1E depict luminescence in supernatants ofHEK293 (FIGS. 1A, 1D, and 1E), HepG2 (FIG. 1B), or 1C1C7 (FIG. 1C) cells24 hours after transfection with circular RNA comprising a Gaussialuciferase expression sequence and various IRES sequences.

FIG. 2 depicts luminescence in supernatants of HEK293 (FIG. 2A), HepG2(FIG. 2B), or 1C1C7 (FIG. 2C) cells 24 hours after transfection withcircular RNA comprising a Gaussia luciferase expression sequence andvarious IRES sequences having different lengths.

FIGS. 3A and 3B depict stability of select IRES constructs in HepG2(FIG. 3A) or 1C1C7 (FIG. 3B) cells over 3 days as measured byluminescence.

FIGS. 4A and 4B depict protein expression from select IRES constructs inJurkat cells, as measured by luminescence from secreted Gaussialuciferase in cell supernatants.

FIGS. 5A and 5B depict stability of select IRES constructs in Jurkatcells over 3 days as measured by luminescence.

FIGS. 6A and 6B depict comparisons of 24 hour luminescence (FIG. 6A) orrelative luminescence over 3 days (FIG. 6B) of modified linear,unpurified circular, or purified circular RNA encoding Gaussialuciferase.

FIGS. 7A, 7B, 7C, 7D, 7E, and 7F depict transcript induction of IFNγ(FIG. 7A), IL-6 (FIG. 7B), IL-2 (FIG. 7C), RIG-I (FIG. 7D), IFN-01 (FIG.7E), and TNFα (FIG. 7F) after electroporation of Jurkat cells withmodified linear, unpurified circular, or purified circular RNA.

FIGS. 8A, 8B, and 8C depict a comparison of luminescence of circular RNAand modified linear RNA encoding Gaussia luciferase in human primarymonocytes (FIG. 8A) and macrophages (FIG. 8B and FIG. 8C).

FIGS. 9A and 9B depict relative luminescence over 3 days (FIG. 9A) insupernatant of primary T cells after transduction with circular RNAcomprising a Gaussia luciferase expression sequence and varying IRESsequences or 24 hour luminescence (FIG. 9B).

FIGS. 10A, 10B, and 10C depict 24 hour luminescence in supernatant ofprimary T cells (FIG. 10A) after transduction with circular RNA ormodified linear RNA comprising a Gaussia luciferase expression sequence,or relative luminescence over 3 days (FIG. 10B), and 24 hourluminescence in PBMCs (FIG. 10C).

FIGS. 11A and 11B depict HPLC chromatograms (FIG. 11A) andcircularization efficiencies (FIG. 11B) of RNA constructs havingdifferent permutation sites.

FIGS. 12A and 12B depict HPLC chromatograms (FIG. 12A) andcircularization efficiencies (FIG. 12B) of RNA constructs havingdifferent introns and/or permutation sites.

FIGS. 13A and 13B depict HPLC chromatograms (FIG. 13A) andcircularization efficiencies (FIG. 13B) of 3 RNA constructs with orwithout homology arms.

FIG. 14 depicts circularization efficiencies of 3 RNA constructs withouthomology arms or with homology arms having various lengths and GCcontent.

FIGS. 15A and 15B depict HPLC chromatograms showing the contribution ofstrong homology arms to improved splicing efficiency, the relationshipbetween circularization efficiency and nicking in select constructs, andcombinations of permutations sites and homology arms hypothesized todemonstrate improved circularization efficiency.

FIG. 16 shows fluorescent images of T cells mock electroporated (left)or electroporated with circular RNA encoding a CAR (right) andco-cultured with Raji cells expressing GFP and firefly luciferase.

FIG. 17 shows bright field (left), fluorescent (center), and overlay(right) images of T cells mock electroporated (top) or electroporatedwith circular RNA encoding a CAR (bottom) and co-cultured with Rajicells expressing GFP and firefly luciferase.

FIG. 18 depicts specific lysis of Raji target cells by T cells mockelectroporated or electroporated with circular RNA encoding differentCAR sequences.

FIGS. 19A and 19B depict luminescence in supernatants of Jurkat cells(left) or resting primary human CD3+ T cells (right) 24 hours aftertransduction with linear or circular RNA comprising a Gaussia luciferaseexpression sequence and varying IRES sequences (FIG. 19A), and relativeluminescence over 3 days (FIG. 19B).

FIGS. 20A, 20B, 20C, 20D, 20E, and 20F depict transcript induction ofIFN-β1 (FIG. 20A), RIG-I (FIG. 20B), IL-2 (FIG. 20C), IL-6 (FIG. 20D),IFNγ (FIG. 20E), and TNFα (FIG. 20F) after electroporation of human CD3+T cells with modified linear, unpurified circular, or purified circularRNA.

FIG. 21 depicts specific lysis of Raji target cells by human primaryCD3+ T cells electroporated with circRNA encoding a CAR as determined bydetection of firefly luminescence (FIG. 21A), and IFNγ transcriptinduction 24 hours after electroporation with different quantities ofcircular or linear RNA encoding a CAR sequence (FIG. 21B).

FIGS. 22A and 22B depict specific lysis of target or non-target cells byhuman primary CD3+ T cells electroporated with circular or linear RNAencoding a CAR at different E:T ratios (FIG. 22A and FIG. 22B) asdetermined by detection of firefly luminescence.

FIG. 23 depicts specific lysis of target cells by human CD3+ T cellselectroporated with RNA encoding a CAR at 1, 3, 5, and 7 days postelectroporation.

FIG. 24 depicts specific lysis of target cells by human CD3+ T cellselectroporated with circular RNA encoding a CD19 or BCMA targeted CAR.

FIG. 25 depicts total Flux of organs harvested from CD-1 mice dosed withcircular RNA encoding FLuc and formulated with 50% Lipid 15 (Table 10b),10% DSPC, 1.5% PEG-DMG, and 38.5% cholesterol.

FIG. 26 shows images highlighting the luminescence of organs harvestedfrom CD-1 mice dosed with circular RNA encoding FLuc and formulated with50% Lipid 15 (Table 10b), 10% DSPC, 1.5% PEG-DMG, and 38.5% cholesterol.

FIG. 27 depicts molecular characterization of Lipids 26 and 27 fromTable 10a. FIG. 27A shows the proton nuclear magnetic resonance (NMR)spectrum of Lipid 26. FIG. 27B shows the retention time of Lipid 26measured by liquid chromatography-mass spectrometry (LC-MS). FIG. 27Cshows the mass spectrum of Lipid 26. FIG. 27D shows the proton NMRspectrum of Lipid 27. FIG. 27E shows the retention time of Lipid 27measured by LC-MS. FIG. 27F shows the mass spectrum of Lipid 27.

FIGS. 28A, 28B, and 28C depict molecular characterization of Lipid22-S14 and its synthetic intermediates. FIG. 28A depicts the NMRspectrum of 2-(tetradecylthio)ethan-1-ol. FIG. 28B depicts the NMRspectrum of 2-(tetradecylthio)ethyl acrylate. FIG. 28C depicts the NMRspectrum of bis(2-(tetradecylthio)ethyl)3,3′-((3-(2-methyl-1H-imidazol-1-yl)propyl) azanediyl)dipropionate(Lipid 22-S14).

FIG. 29 depicts the NMR spectrum of bis(2-(tetradecylthio)ethyl)3,3′-((3-(1H-imidazol-1-yl)propyl) azanediyl)dipropionate (Lipid93-S14).

FIGS. 30A, 30B, and 30C depict molecular characterization ofheptadecan-9-yl8-((3-(2-methyl-1H-imidazol-1-yl)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate(Lipid 54 from Table 10a). FIG. 30A shows the proton NMR spectrum ofLipid 54. FIG. 30B shows the retention time of Lipid 54 measured byLC-MS. FIG. 30C shows the mass spectrum of Lipid 54.

FIGS. 31A, 31B, and 31C depict molecular characterization ofheptadecan-9-yl8-((3-(1H-imidazol-1-yl)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate(Lipid 53 from Table 10a). FIG. 31A shows the proton NMR spectrum ofLipid 53. FIG. 31B shows the retention time of Lipid 53 measured byLC-MS. FIG. 31C shows the mass spectrum of Lipid 53.

FIG. 32A depicts total flux of spleen and liver harvested from CD-1 micedosed with circular RNA encoding firefly luciferase (FLuc) andformulated with ionizable lipid of interest, DSPC, cholesterol, andDSPE-PEG 2000 (Avanti Polar Lipids Inc.) at a weight ratio of 16:1:4:1or 62:4:33:1 molar ratio. FIG. 32B depicts average radiance forbiodistribution of protein expression.

FIG. 33A depicts images highlighting the luminescence of organsharvested from CD-1 mice dosed with circular RNA encoding FLuc andformulated with ionizable Lipid 22-S14, DSPC, cholesterol, and DSPE-PEG2000 (Avanti Polar Lipids Inc.) at a weight ratio of 16:1:4:1 or62:4:33:1 molar ratio. FIG. 33B depicts whole body IVIS images of CD-1mice dosed with circular RNA encoding FLuc and formulated with ionizableLipid 22-S14, DSPC, cholesterol, and DSPE-PEG 2000 (Avanti Polar LipidsInc.) at a weight ratio of 16:1:4:1 or 62:4:33:1 molar ratio.

FIG. 34A depicts images highlighting the luminescence of organsharvested from CD-1 mice dosed with circular RNA encoding FLuc andformulated with ionizable Lipid 93-S14, DSPC, cholesterol, and DSPE-PEG2000 (Avanti Polar Lipids Inc.) at a weight ratio of 16:1:4:1 or62:4:33:1 molar ratio. FIG. 34B depicts whole body IVIS images of CD-1mice dosed with circular RNA encoding FLuc and formulated with ionizableLipid 93-S14, DSPC, cholesterol, and DSPE-PEG 2000 (Avanti Polar LipidsInc.) at a weight ratio of 16:1:4:1 or 62:4:33:1 molar ratio.

FIG. 35A depicts images highlighting the luminescence of organsharvested from CD-1 mice dosed with circular RNA encoding FLuc andformulated with ionizable Lipid 26 from Table 10a, DSPC, cholesterol,and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) at a weight ratio of16:1:4:1 or 62:4:33:1 molar ratio. FIG. 35B depicts whole body IVISimages of CD-1 mice dosed with circular RNA encoding FLuc and formulatedwith ionizable Lipid 26, DSPC, cholesterol, and DSPE-PEG 2000 (AvantiPolar Lipids Inc.) at a weight ratio of 16:1:4:1 or 62:4:33:1 molarratio.

FIGS. 36A, 36B, 36C, and 36D depict images highlighting the luminescenceof organs harvested from c57BL/6J mice dosed with circular RNA encodingFLuc and encapsulated in lipid nanoparticles formed with Lipid 15 fromTable 10b (FIG. 36A), Lipid 53 from Table 10a (FIG. 36B), or Lipid 54from Table 10a (FIG. 36C). PBS was used as control (FIG. 36D).

FIGS. 37A and 37B depict relative luminescence in the lysates of humanPBMCs after 24-hour incubation with testing lipid nanoparticlescontaining circular RNA encoding firefly luciferase.

FIGS. 38A and 38B show the expression of GFP (FIG. 38A) and CD19 CAR(FIG. 38B) in human PBMCs after incubating with testing lipidnanoparticle containing circular RNA encoding either GFP or CD19 CAR.

FIG. 39 depicts the expression of an anti-murine CD19 CAR in 1C1C7 cellslipotransfected with circular RNA comprising an anti-murine CD19 CARexpression sequence and varying IRES sequences.

FIG. 40 shows the cytotoxicity of an anti-murine CD19 CAR to murine Tcells. The CD19 CAR is encoded by and expressed from a circular RNA,which is electroporated into the murine T cells.

FIGS. 41A, 41B and 41C depict the B cell counts in peripheral blood(FIGS. 41A and 41B) or spleen (FIG. 41C) in C57BL/6J mice injected everyother day with testing lipid nanoparticles encapsulating a circular RNAencoding an anti-murine CD19 CAR.

FIGS. 42A and 42B compares the expression level of an anti-human CD19CAR expressed from a circular RNA with that expressed from a linearmRNA.

FIGS. 43A and 43B compares the cytotoxic effect of an anti-human CD19CAR expressed from a circular RNA with that expressed from a linear mRNA

FIG. 44 depicts the cytotoxicity of two CARs (anti-human CD19 CAR andanti-human BCMA CAR) expressed from a single circular RNA in T cells.

FIG. 45A shows representative FACS plots with frequencies of tdTomatoexpression in various spleen immune cell subsets following treatmentwith LNPs formed with Lipid 27 or 26 from Table 10a or Lipid 15 fromTable 10b. FIG. 45B shows the quantification of the proportion ofmyeloid cells, B cells, and T cells expressing tdTomato (mean+std. dev.,n=3), equivalent to the proportion of each cell population successfullytransfected with Cre circular RNA. FIG. 45C illustrates the proportionof additional splenic immune cell populations, including NK cells,classical monocytes, nonclassical monocytes, neutrophils, and dendriticcells, expressing tdTomato after treatment with Lipids 27 and 26(mean+std. dev., n=3).

FIG. 46A depicts an exemplary RNA construct design with built-in polyAsequences in the introns. FIG. 46B shows the chromatography trace ofunpurified circular RNA. FIG. 46C shows the chromatography trace ofaffinity-purified circular RNA. FIG. 46D shows the immunogenicity of thecircular RNAs prepared with varying IVT conditions and purificationmethods. (Commercial=commercial IVT mix; Custom=customerized IVT mix;Aff=affinity purification; Enz=enzyme purification; GMP:GTP ratio=8,12.5, or 13.75).

FIG. 47A depicts an exemplary RNA construct design with a dedicatedbinding sequence as an alternative to polyA for hybridizationpurification. FIG. 47B shows the chromatography trace of unpurifiedcircular RNA. FIG. 47C shows the chromatography trace ofaffinity-purified circular RNA.

FIG. 48A shows the chromatography trace of unpurified circular RNAencoding dystrophin. FIG. 48B shows the chromatography trace ofenzyme-purified circular RNA encoding dystrophin.

FIG. 49 compares the expression (FIG. 49A) and stability (FIG. 49B) ofpurified circRNAs with different 5′ spacers between the 3′ intronfragment/5′ internal duplex region and the IRES in Jurkat cells.(AC=only A and C were used in the spacer sequence; UC=only U and C wereused in the spacer sequence.)

FIG. 50 shows luminescence expression levels and stability of expressionin primary T cells from circular RNAs containing the original ormodified IRES elements indicated.

FIG. 51 shows luminescence expression levels and stability of expressionin HepG2 cells from circular RNAs containing the original or modifiedIRES elements indicated.

FIG. 52 shows luminescence expression levels and stability of expressionin 1C1C7 cells from circular RNAs containing the original or modifiedIRES elements indicated.

FIG. 53 shows luminescence expression levels and stability of expressionin HepG2 cells from circular RNAs containing IRES elements withuntranslated regions (UTRs) inserted or hybrid IRES elements. “Scr”means Scrambled, which was used as a control.

FIG. 54 shows luminescence expression levels and stability of expressionin 1C1C7 cells from circular RNAs containing an IRES and variable stopcodon cassettes operably linked to a Gaussia luciferase coding sequence.

FIG. 55 shows luminescence expression levels and stability of expressionin 1C1C7 cells from circular RNAs containing an IRES and variableuntranslated regions (UTRs) inserted before the start codon of agaussian luciferase coding sequence.

FIG. 56 shows expression levels of human erythropoietin (hEPO) in Huh7cells from circular RNAs containing two miR-122 target sites downstreamfrom the hEPO coding sequence.

DETAILED DESCRIPTION

Provided herein are pharmaceutical compositions and transfer vehicles,e.g., lipid nanoparticles, comprising circular RNA. The circular RNAprovided herein may be delivered and/or targeted to a cell in a transfervehicle, e.g., a nanoparticle, or a composition comprising a transfervehicle. In some embodiments, the circular RNA may also be delivered toa subject in a transfer vehicle or a composition comprising a transfervehicle. In some embodiments, the transfer vehicle is a nanoparticle. Insome embodiments, the nanoparticle is a lipid nanoparticle, a polymericcore-shell nanoparticle, or a biodegradable nanoparticle. In someembodiments, the nanoparticle is a lipid nanoparticle. In someembodiments, the transfer vehicle comprises one or more ionizablelipids, PEG modified lipids, helper lipids, and/or structural lipids.

In some embodiments, a transfer vehicle encapsulates circular RNA andcomprises an ionizable lipid, a structural lipid, and a PEG-modifiedlipid. In some embodiments, a transfer vehicle encapsulates circular RNAand comprises an ionizable lipid, a structural lipid, a PEG-modifiedlipid, and a helper lipid.

In some embodiments, the transfer vehicle comprises an ionizable lipiddescribed herein. In some embodiments, the transfer vehicle comprises anionizable lipid shown in any one of Tables 1-10, 10a, 10b, 11-15, and15b. In some embodiments, the transfer vehicle comprises an ionizablelipid shown in Table 10a.

In some embodiments, the RNA in a transfer vehicle is at least about80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%,99.9%, or more circular RNA. In some embodiments, less than 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% of loadedRNA is on or associated with a transfer vehicle exterior surface.

In some embodiments, the transfer vehicle is capable of binding to APOE.In some embodiments, the surface of the transfer vehicle comprises APOEbinding sites. In some embodiments, the surface of the transfer vehicleis substantially free of APOE binding sites. In some embodiments, atransfer vehicle interacts with APOE less than an equivalent transfervehicle loaded with linear RNA. In some embodiments, APOE interactionmay be measured by comparing nanoparticle uptake in cells in APOdepleted serum or APO complement serum.

Without wishing to be bound by theory, it is contemplated that transfervehicles comprising APOE binding sites deliver circular RNAs moreefficiently to the liver. Accordingly, in some embodiments, the transfervehicle comprising the ionizable lipids described herein and loaded withcircular RNA substantially comprises APOE binding sites on the transfervehicle surface, thereby delivering the circular RNA to the liver at ahigher efficiency compared to a transfer vehicle substantially lackingAPOE binding sites on the surface. In some embodiments, the transfervehicle comprising the ionizable lipids described herein and loaded withcircular RNA substantially lacks APOE binding sites on the transfervehicle surface, thereby delivering the circular RNA to the liver at alower efficiency compared to a transfer vehicle comprising APOE bindingsites on the surface.

In some embodiments, the transfer vehicle delivers, or is capable ofdelivering, circular RNA to the spleen. In some embodiments, a circularRNA encodes a therapeutic protein. In some embodiments, at least about10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 99% of the total therapeutic protein expressed inthe subject is expressed in the spleen. In some embodiments, moretherapeutic protein is expressed in the spleen than in the liver (e.g.,2×, 3×, 4×, or 5× more). In some embodiments, the lipid nanoparticle hasan ionizable lipid:phosphate ratio of 3-7. In some embodiments, thelipid nanoparticle has an ionizable lipid:phosphate ratio of 4-6. Insome embodiments, the lipid nanoparticle has an ionizablelipid:phosphate ratio of 4.5. In some embodiments, the lipidnanoparticle has an nitrogen:phosphate (N:P) ratio of 3-6. In someembodiments, the lipid nanoparticle has an N:P ratio of 5-6. In someembodiments, the lipid nanoparticle has an N:P ratio of 5.7. In someembodiments, expression of a nonsecreted protein may be measured usingan ELISA, normalizing to tissue weight.

Without wishing to be bound by theory, it is thought that transfervehicles described herein shield encapsulated circular RNA fromdegradation and provide for effective delivery of circular RNA to targetcells in vivo and in vitro.

Embodiments of the present disclosure provide lipid compositionsdescribed according to the respective molar ratios of the componentlipids in the formulation. In one embodiment, the mol-% of the ionizablelipid may be from about 10 mol-% to about 80 mol-%. In one embodiment,the mol-% of the ionizable lipid may be from about 20 mol-% to about 70mol-%. In one embodiment, the mol-% of the ionizable lipid may be fromabout 30 mol-% to about 60 mol-%. In one embodiment, the mol-% of theionizable lipid may be from about 35 mol-% to about 55 mol-%. In oneembodiment, the mol-% of the ionizable lipid may be from about 40 mol-%to about 50 mol-%. In some embodiments, the ionizable lipid mol-% of thetransfer vehicle batch will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or±2.5% of the target mol-%. In certain embodiments, transfer vehicleinter-lot variability will be less than 15%, less than 10% or less than5%.

In one embodiment, the mol-% of the helper lipid may be from about 1mol-% to about 50 mol-%. In one embodiment, the mol-% of the helperlipid may be from about 2 mol-% to about 45 mol-%. In one embodiment,the mol-% of the helper lipid may be from about 3 mol-% to about 40mol-%. In one embodiment, the mol-% of the helper lipid may be fromabout 4 mol-% to about 35 mol-%. In one embodiment, the mol-% of thehelper lipid may be from about 5 mol-% to about 30 mol-%. In oneembodiment, the mol-% of the helper lipid may be from about 10 mol-% toabout 20 mol-%. In some embodiments, the helper lipid mol-% of thetransfer vehicle batch will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or±2.5% of the target mol-%.

In one embodiment, the mol-% of the structural lipid may be from about10 mol-% to about 80 mol-%. In one embodiment, the mol-% of thestructural lipid may be from about 20 mol-% to about 70 mol-%. In oneembodiment, the mol-% of the structural lipid may be from about 30 mol-%to about 60 mol-%. In one embodiment, the mol-% of the structural lipidmay be from about 35 mol-% to about 55 mol-%. In one embodiment, themol-% of the structural lipid may be from about 40 mol-% to about 50mol-%. In some embodiments, the structural lipid mol-% of the transfervehicle batch will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of thetarget mol-%.

In one embodiment, the mol-% of the PEG modified lipid may be from about0.1 mol-% to about 10 mol-%. In one embodiment, the mol-% of the PEGmodified lipid may be from about 0.2 mol-% to about 5 mol-%. In oneembodiment, the mol-% of the PEG modified lipid may be from about 0.5mol-% to about 3 mol-%. In one embodiment, the mol-% of the PEG modifiedlipid may be from about 1 mol-% to about 2 mol-%. In one embodiment, themol-% of the PEG modified lipid may be about 1.5 mol-%. In someembodiments, the PEG modified lipid mol-% of the transfer vehicle batchwill be ±30%, ±25%, +20%, ±15%, ±10%, ±5%, or ±2.5% of the target mol-%.

Also contemplated are pharmaceutical compositions, and in particulartransfer vehicles, that comprise one or more of the compounds disclosedherein. In certain embodiments, such transfer vehicles comprise one ormore of PEG-modified lipids, an ionizable lipid, a helper lipid, and/ora structural lipid disclosed herein. Also contemplated are transfervehicles that comprise one or more of the compounds disclosed herein andthat further comprise one or more additional lipids. In certainembodiments, such transfer vehicles are loaded with or otherwiseencapsulate circular RNA.

Transfer vehicles of the invention encapsulate circular RNA. In certainembodiments, the polynucleotides encapsulated by the compounds orpharmaceutical and liposomal compositions of the invention include RNAencoding a protein or enzyme (e.g., circRNA encoding, for example,phenylalanine hydroxylase (PAH)). The present invention contemplates theuse of such polynucleotides as a therapeutic that is capable of beingexpressed by target cells to thereby facilitate the production (and incertain instances, the excretion) of a functional enzyme or protein asdisclosed by such target cells, for example, in InternationalApplication No. PCT/US2010/058457 and in U.S. Provisional ApplicationNo. 61/494,881, filed Jun. 8, 2011, the teachings of which are bothincorporated herein by reference in their entirety. For example, incertain embodiments, upon the expression of one or more polynucleotidesby target cells, the production of a functional enzyme or protein inwhich a subject is deficient (e.g., a urea cycle enzyme or an enzymeassociated with a lysosomal storage disorder) may be observed. Asanother example, circular RNA encapsulated by a transfer vehicle mayencode one or both polypeptide chains of a T cell receptor protein orencode a chimeric antigen receptor (CAR).

Also provided herein are methods of treating a disease in a subject byadministering an effective amount of a composition comprising circularRNA encoding a functional protein and a transfer vehicle describedherein to the subject. In some embodiments, the circular RNA isencapsulated within the transfer vehicle. In certain embodiments, suchmethods may enhance (e.g., increase) the expression of a polynucleotideand/or increase the production and secretion of a functional polypeptideproduct in one or more target cells and tissues (e.g., immune cells orhepatocytes). Generally, such methods comprise contacting the targetcells with one or more compounds and/or transfer vehicles that compriseor otherwise encapsulate the circRNA.

In certain embodiments, the transfer vehicles (e.g., lipidnanoparticles) are formulated based in part upon their ability tofacilitate the transfection (e.g., of a circular RNA) of a target cell.In another embodiment, the transfer vehicles (e.g., lipid nanoparticles)may be selected and/or prepared to optimize delivery of circular RNA toa target cell, tissue or organ. For example, if the target cell is ahepatocyte, or if the target organ is the spleen, the properties of thepharmaceutical and/or liposomal compositions (e.g., size, charge and/orpH) may be optimized to effectively deliver such composition (e.g.,lipid nanoparticles) to the target cell or organ, reduce immuneclearance and/or promote retention in the target cell or organ.Alternatively, if the target tissue is the central nervous system, theselection and preparation of the transfer vehicle must considerpenetration of, and retention within. the blood brain barrier and/or theuse of alternate means of directly delivering such compositions (e.g.,lipid nanoparticles) to such target tissue (e.g., viaintracerebrovascular administration). In certain embodiments, thetransfer vehicles may be combined with agents that facilitate thetransfer of encapsulated materials across the blood brain barrier (e.g.,agents which disrupt or improve the permeability of the blood brainbarrier and thereby enhance the transfer of circular RNA to the targetcells). While the transfer vehicles described herein (e.g., lipidnanoparticles) can facilitate introduction of circRNA into target cells,the addition of polycations (e.g., poly L-lysine and protamine) to, forexample, one or more of the lipid nanoparticles that comprise thepharmaceutical compositions as a copolymer can also facilitate, and insome instances markedly enhance, the transfection efficiency of severaltypes of transfer vehicles by 2-28 fold in a number of cell lines bothin vitro and in vivo (See, N. J. Caplen, et al., Gene Ther. 1995; 2:603; S. Li, et al., Gene Ther. 1997; 4, 891). In some embodiments, atarget cell is an immune cell. In some embodiments, a target cell is a Tcell.

In certain embodiments, the transfer vehicles described herein (e.g.,lipid nanoparticles) are prepared by combining multiple lipid components(e.g., one or more of the compounds disclosed herein) with one or morepolymer components. For example, a lipid nanoparticle may be preparedusing HGT4003, DOPE, cholesterol and DMG-PEG2000. A lipid nanoparticlemay be comprised of additional lipid combinations in various ratios,including for example, HGT4001, DOPE and DMG-PEG2000. The selection ofionizable lipids, helper lipids, structural lipids, and/or PEG-modifiedlipids which comprise the lipid nanoparticles, as well as the relativemolar ratio of such lipids to each other, is based upon thecharacteristics of the selected lipid(s), the nature of the intendedtarget cells or tissues and the characteristics of the materials orpolynucleotides to be delivered by the lipid nanoparticle. Additionalconsiderations include, for example, the saturation of the alkyl chain,as well as the size, charge, pH, pKa, fusogenicity and toxicity of theselected lipid(s).

Transfer vehicles described herein can allow the encapsulatedpolynucleotide to reach the target cell or may preferentially allow theencapsulated polynucleotide to reach the target cells or organs on adiscriminatory basis (e.g., the transfer vehicles may concentrate in theliver or spleen of a subject to which such transfer vehicles areadministered). Alternatively, the transfer vehicles may limit thedelivery of encapsulated polynucleotides to other non-targeted cells ororgans where the presence of the encapsulated polynucleotides may beundesirable or of limited utility.

Loading or encapsulating a polynucleotide, e.g., circRNA, into atransfer vehicle may serve to protect the polynucleotide from anenvironment (e.g., serum) which may contain enzymes or chemicals thatdegrade such polynucleotides and/or systems or receptors that cause therapid excretion of such polynucleotides. Accordingly, in someembodiments, the compositions described herein are capable of enhancingthe stability of the encapsulated polynucleotide(s), particularly withrespect to the environments into which such polynucleotides will beexposed.

In certain embodiments, provided herein is a vector for making circularRNA, the vector comprising a 5′ duplex forming region, a 3′ group Iintron fragment, optionally a first spacer, an Internal Ribosome EntrySite (IRES), an expression sequence, optionally a second spacer, a 5′group I intron fragment, and a 3′ duplex forming region. In someembodiments, these elements are positioned in the vector in the aboveorder. In some embodiments, the vector further comprises an internal 5′duplex forming region between the 3′ group I intron fragment and theIRES and an internal 3′ duplex forming region between the expressionsequence and the 5′ group I intron fragment. In some embodiments, theinternal duplex forming regions are capable of forming a duplex betweeneach other but not with the external duplex forming regions. In someembodiments, the internal duplex forming regions are part of the firstand second spacers. Additional embodiments include circular RNApolynucleotides, including circular RNA polynucleotides made using thevectors provided herein, compositions comprising such circular RNA,cells comprising such circular RNA, methods of using and making suchvectors, circular RNA, compositions and cells.

In some embodiments, provided herein are methods comprisingadministration of circular RNA polynucleotides provided herein intocells for therapy or production of useful proteins, such as PAH. In someembodiments, the method is advantageous in providing the production of adesired polypeptide inside eukaryotic cells with a longer half-life thanlinear RNA, due to the resistance of the circular RNA to ribonucleases.

Circular RNA polynucleotides lack the free ends necessary forexonuclease-mediated degradation, causing them to be resistant toseveral mechanisms of RNA degradation and granting extended half-liveswhen compared to an equivalent linear RNA. Circularization may allow forthe stabilization of RNA polynucleotides that generally suffer fromshort half-lives and may improve the overall efficacy of exogenous mRNAin a variety of applications. In an embodiment, the half-life of thecircular RNA polynucleotides provided herein in eukaryotic cells (e.g.,mammalian cells, such as human cells) is at least 20 hours (e.g., atleast 80 hours).

1. Definitions

As used herein, the terms “circRNA” or “circular polyribonucleotide” or“circular RNA” or “oRNA” are used interchangeably and refers to apolyribonucleotide that forms a circular structure through covalentbonds.

As used herein, the term “3′ group I intron fragment” refers to asequence with 75% or higher similarity to the 3′-proximal end of anatural group I intron including the splice site dinucleotide andoptionally a stretch of natural exon sequence.

As used herein, the term “5′ group I intron fragment” refers to asequence with 75% or higher similarity to the 5′-proximal end of anatural group I intron including the splice site dinucleotide andoptionally a stretch of natural exon sequence.

As used herein, the term “permutation site” refers to the site in agroup I intron where a cut is made prior to permutation of the intron.This cut generates 3′ and 5′ group I intron fragments that are permutedto be on either side of a stretch of precursor RNA to be circularized.

As used herein, the term “splice site” refers to a dinucleotide that ispartially or fully included in a group I intron and between which aphosphodiester bond is cleaved during RNA circularization.

As used herein, the term “therapeutic protein” refers to any proteinthat, when administered to a subject directly or indirectly in the formof a translated nucleic acid, has a therapeutic, diagnostic, and/orprophylactic effect and/or elicits a desired biological and/orpharmacological effect.

As used herein, the term “immunogenic” refers to a potential to inducean immune response to a substance. An immune response may be inducedwhen an immune system of an organism or a certain type of immune cellsis exposed to an immunogenic substance. The term “non-immunogenic”refers to a lack of or absence of an immune response above a detectablethreshold to a substance. No immune response is detected when an immunesystem of an organism or a certain type of immune cells is exposed to anon-immunogenic substance. In some embodiments, a non-immunogeniccircular polyribonucleotide as provided herein, does not induce animmune response above a pre-determined threshold when measured by animmunogenicity assay. In some embodiments, no innate immune response isdetected when an immune system of an organism or a certain type ofimmune cells is exposed to a non-immunogenic circular polyribonucleotideas provided herein. In some embodiments, no adaptive immune response isdetected when an immune system of an organism or a certain type ofimmune cell is exposed to a non-immunogenic circular polyribonucleotideas provided herein.

As used herein, the term “circularization efficiency” refers to ameasurement of resultant circular polyribonucleotide as compared to itslinear starting material.

As used herein, the term “translation efficiency” refers to a rate oramount of protein or peptide production from a ribonucleotidetranscript. In some embodiments, translation efficiency can be expressedas amount of protein or peptide produced per given amount of transcriptthat codes for the protein or peptide.

The term “nucleotide” refers to a ribonucleotide, a deoxyribonucleotide,a modified form thereof, or an analog thereof. Nucleotides includespecies that comprise purines, e.g., adenine, hypoxanthine, guanine, andtheir derivatives and analogs, as well as pyrimidines, e.g., cytosine,uracil, thymine, and their derivatives and analogs. Nucleotide analogsinclude nucleotides having modifications in the chemical structure ofthe base, sugar and/or phosphate, including, but not limited to,5′-position pyrimidine modifications, 8′-position purine modifications,modifications at cytosine exocyclic amines, and substitution of5-bromo-uracil; and 2′-position sugar modifications, including but notlimited to, sugar-modified ribonucleotides in which the 2′-OH isreplaced by a group such as an H, OR, R, halo, SH, SR, NH₂, NHR, NR₂, orCN, wherein R is an alkyl moiety as defined herein. Nucleotide analogsare also meant to include nucleotides with bases such as inosine,queuosine, xanthine; sugars such as 2′-methyl ribose; non-naturalphosphodiester linkages such as methylphosphonate, phosphorothioate andpeptide linkages. Nucleotide analogs include 5-methoxyuridine,1-methylpseudouridine, and 6-methyladenosine.

The term “nucleic acid” and “polynucleotide” are used interchangeablyherein to describe a polymer of any length, e.g., greater than about 2bases, greater than about 10 bases, greater than about 100 bases,greater than about 500 bases, greater than 1000 bases, or up to about10,000 or more bases, composed of nucleotides, e.g.,deoxyribonucleotides or ribonucleotides, and may be producedenzymatically or synthetically (e.g., as described in U.S. Pat. No.5,948,902 and the references cited therein), which can hybridize withnaturally occurring nucleic acids in a sequence specific manneranalogous to that of two naturally occurring nucleic acids, e.g., canparticipate in Watson-Crick base pairing interactions. Naturallyoccurring nucleic acids are comprised of nucleotides including guanine,cytosine, adenine, thymine, and uracil (G, C, A, T, and U respectively).

The terms “ribonucleic acid” and “RNA” as used herein mean a polymercomposed of ribonucleotides.

The terms “deoxyribonucleic acid” and “DNA” as used herein mean apolymer composed of deoxyribonucleotides.

“Isolated” or “purified” generally refers to isolation of a substance(for example, in some embodiments, a compound, a polynucleotide, aprotein, a polypeptide, a polynucleotide composition, or a polypeptidecomposition) such that the substance comprises a significant percent(e.g., greater than 1%, greater than 2%, greater than 5%, greater than10%, greater than 20%, greater than 50%, or more, usually up to about90%-100%) of the sample in which it resides. In certain embodiments, asubstantially purified component comprises at least 50%, 80%-85%, or90%-95% of the sample. Techniques for purifying polynucleotides andpolypeptides of interest are well-known in the art and include, forexample, ion-exchange chromatography, affinity chromatography andsedimentation according to density. Generally, a substance is purifiedwhen it exists in a sample in an amount, relative to other components ofthe sample, that is more than as it is found naturally.

The terms “duplexed,” “double-stranded,” or “hybridized” as used hereinrefer to nucleic acids formed by hybridization of two single strands ofnucleic acids containing complementary sequences. In most cases, genomicDNA is double-stranded. Sequences can be fully complementary orpartially complementary.

As used herein, “unstructured” with regard to RNA refers to an RNAsequence that is not predicted by the RNAFold software or similarpredictive tools to form a structure (e.g., a hairpin loop) with itselfor other sequences in the same RNA molecule. In some embodiments,unstructured RNA can be functionally characterized using nucleaseprotection assays.

As used herein, “structured” with regard to RNA refers to an RNAsequence that is predicted by the RNAFold software or similar predictivetools to form a structure (e.g., a hairpin loop) with itself or othersequences in the same RNA molecule.

As used herein, two “duplex forming regions,” “homology arms,” or“homology regions,” may be any two regions that are thermodynamicallyfavored to cross-pair in a sequence specific interaction. In someembodiments, two duplex forming regions, homology arms, or homologyregions, share a sufficient level of sequence identity to one another'sreverse complement to act as substrates for a hybridization reaction. Asused herein polynucleotide sequences have “homology” when they areeither identical or share sequence identity to a reverse complement or“complementary” sequence. The percent sequence identity between ahomology region and a counterpart homology region's reverse complementcan be any percent of sequence identity that allows for hybridization tooccur. In some embodiments, an internal duplex forming region of aninventive polynucleotide is capable of forming a duplex with anotherinternal duplex forming region and does not form a duplex with anexternal duplex forming region.

Linear nucleic acid molecules are said to have a “5′-terminus” (5′ end)and a “3′-terminus” (3′ end) because nucleic acid phosphodiesterlinkages occur at the 5′ carbon and 3′ carbon of the sugar moieties ofthe substituent mononucleotides. The end nucleotide of a polynucleotideat which a new linkage would be to a 5′ carbon is its 5′ terminalnucleotide. The end nucleotide of a polynucleotide at which a newlinkage would be to a 3′ carbon is its 3′ terminal nucleotide. Aterminal nucleotide, as used herein, is the nucleotide at the endposition of the 3′- or 5′-terminus

“Transcription” means the formation or synthesis of an RNA molecule byan RNA polymerase using a DNA molecule as a template. The invention isnot limited with respect to the RNA polymerase that is used fortranscription. For example, in some embodiments, a T7-type RNApolymerase can be used.

“Translation” means the formation of a polypeptide molecule by aribosome based upon an RNA template.

It is to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting. As used in this specification and the appended claims,the singular forms “a,” “an,” and “the” include plural referents unlessthe content clearly dictates otherwise. Thus, for example, reference to“a cell” includes combinations of two or more cells, or entire culturesof cells; reference to “a polynucleotide” includes, as a practicalmatter, many copies of that polynucleotide. Unless specifically statedor obvious from context, as used herein, the term “or” is understood tobe inclusive. Unless defined herein and below in the reminder of thespecification, all technical and scientific terms used herein have thesame meaning as commonly understood by one of ordinary skill in the artto which the invention pertains.

Unless specifically stated or obvious from context, as used herein, theterm “about,” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. “About” canbe understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%,0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%,0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% of the stated value. Unlessotherwise clear from the context, all numerical values provided hereinare modified by the term “about.”

As used herein, the term “encode” refers broadly to any process wherebythe information in a polymeric macromolecule is used to direct theproduction of a second molecule that is different from the first. Thesecond molecule may have a chemical structure that is different from thechemical nature of the first molecule.

By “co-administering” is meant administering a therapeutic agentprovided herein in conjunction with one or more additional therapeuticagents sufficiently close in time such that the therapeutic agentprovided herein can enhance the effect of the one or more additionaltherapeutic agents, or vice versa.

The terms “treat,” and “prevent” as well as words stemming therefrom, asused herein, do not necessarily imply 100% or complete treatment orprevention. Rather, there are varying degrees of treatment or preventionof which one of ordinary skill in the art recognizes as having apotential benefit or therapeutic effect. The treatment or preventionprovided by the method disclosed herein can include treatment orprevention of one or more conditions or symptoms of the disease. Also,for purposes herein, “prevention” can encompass delaying the onset ofthe disease, or a symptom or condition thereof.

As used herein, the term “expression sequence” refers to a nucleic acidsequence that encodes a product, e.g., a peptide or polypeptide,regulatory nucleic acid, or non-coding nucleic acid. An exemplaryexpression sequence that codes for a peptide or polypeptide can comprisea plurality of nucleotide triads, each of which can code for an aminoacid and is termed as a “codon”.

As used herein, a “spacer” refers to a region of a polynucleotidesequence ranging from 1 nucleotide to hundreds or thousands ofnucleotides separating two other elements along a polynucleotidesequence. The sequences can be defined or can be random. A spacer istypically non-coding. In some embodiments, spacers include duplexforming regions.

As used herein, “splice site” refers to the dinucleotide ordinucleotides between which cleavage of the phosphodiester bond occursduring a splicing reaction. A “5′ splice site” refers to the natural 5′dinucleotide of the intron e.g., group I intron, while a “3′ splicesite” refers to the natural 3′ dinucleotide of the intron.

As used herein, an “internal ribosome entry site” or “IRES” refers to anRNA sequence or structural element ranging in size from 10 nt to 1000 ntor more, capable of initiating translation of a polypeptide in theabsence of a typical RNA cap structure. An IRES is typically about 500nt to about 700 nt in length.

As used herein, a “miRNA site” refers to a stretch of nucleotides withina polynucleotide that is capable of forming a duplex with at least 8nucleotides of a natural miRNA sequence.

As used herein, an “endonuclease site” refers to a stretch ofnucleotides within a polynucleotide that is capable of being recognizedand cleaved by an endonuclease protein.

As used herein, “bicistronic RNA” refers to a polynucleotide thatincludes two expression sequences coding for two distinct proteins.These expression sequences can be separated by a nucleotide sequenceencoding a cleavable peptide such as a protease cleavage site. They canalso be separated by a ribosomal skipping element.

As used herein, the term“ribosomal skipping element” refers to anucleotide sequence encoding a short peptide sequence capable of causinggeneration of two peptide chains from translation of one RNA molecule.While not wishing to be bound by theory, it is hypothesized thatribosomal skipping elements function by (1) terminating translation ofthe first peptide chain and re-initiating translation of the secondpeptide chain; or (2) cleavage of a peptide bond in the peptide sequenceencoded by the ribosomai skipping element by an intrinsic proteaseactivity of the encoded peptide, or by another protease in theenvironment (e.g., cytosol).

As used herein, the term “co-formulate” refers to a nanoparticleformulation comprising two or more nucleic acids or a nucleic acid andother active drug substance. Typically, the ratios are equimolar ordefined in the ratiometric amount of the two or more nucleic acids orthe nucleic acid and other active drug substance.

As used herein, “transfer vehicle” includes any of the standardpharmaceutical carriers, diluents, excipients, and the like, which aregenerally intended for use in connection with the administration ofbiologically active agents, including nucleic acids.

As used herein, the phrase “lipid nanoparticle” refers to a transfervehicle comprising one or more lipids (e.g., in some embodiments,cationic lipids, non-cationic lipids, and PEG-modified lipids).

As used herein, the phrase “ionizable lipid” refers to any of a numberof lipid species that carry a net positive charge at a selected pH, suchas physiological pH 4 and a neutral charge at other pHs such asphysiological pH 7.

In some embodiments, a lipid, e.g., an ionizable lipid, disclosed hereincomprises one or more cleavable groups. The terms “cleave” and“cleavable” are used herein to mean that one or more chemical bonds(e.g., one or more of covalent bonds, hydrogen-bonds, van der Waals'forces and/or ionic interactions) between atoms in or adjacent to thesubject functional group are broken (e.g., hydrolyzed) or are capable ofbeing broken upon exposure to selected conditions (e.g., upon exposureto enzymatic conditions). In certain embodiments, the cleavable group isa disulfide functional group, and in particular embodiments is adisulfide group that is capable of being cleaved upon exposure toselected biological conditions (e.g., intracellular conditions). Incertain embodiments, the cleavable group is an ester functional groupthat is capable of being cleaved upon exposure to selected biologicalconditions. For example, the disulfide groups may be cleavedenzymatically or by a hydrolysis, oxidation or reduction reaction. Uponcleavage of such disulfide functional group, the one or more functionalmoieties or groups (e.g., one or more of a head-group and/or atail-group) that are bound thereto may be liberated. Exemplary cleavablegroups may include, but are not limited to, disulfide groups, estergroups, ether groups, and any derivatives thereof (e.g., alkyl and arylesters). In certain embodiments, the cleavable group is not an estergroup or an ether group. In some embodiments, a cleavable group is bound(e.g., bound by one or more of hydrogen-bonds, van der Waals' forces,ionic interactions and covalent bonds) to one or more functionalmoieties or groups (e.g., at least one head-group and at least onetail-group). In certain embodiments, at least one of the functionalmoieties or groups is hydrophilic (e.g., a hydrophilic head-groupcomprising one or more of imidazole, guanidinium, amino, imine, enamine,optionally-substituted alkyl amino and pyridyl).

As used herein, the term “hydrophilic” is used to indicate inqualitative terms that a functional group is water-preferring, andtypically such groups are water-soluble. For example, disclosed hereinare compounds that comprise a cleavable disulfide (S—S) functional groupbound to one or more hydrophilic groups (e.g., a hydrophilichead-group), wherein such hydrophilic groups comprise or are selectedfrom the group consisting of imidazole, guanidinium, amino, imine,enamine, an optionally-substituted alkyl amino (e.g., an alkyl aminosuch as dimethylamino) and pyridyl.

In certain embodiments, at least one of the functional groups ofmoieties that comprise the compounds disclosed herein is hydrophobic innature (e.g., a hydrophobic tail-group comprising a naturally occurringlipid such as cholesterol). As used herein, the term “hydrophobic” isused to indicate in qualitative terms that a functional group iswater-avoiding, and typically such groups are not water soluble. Forexample, disclosed herein are compounds that comprise a cleavablefunctional group (e.g., a disulfide (S—S) group) bound to one or morehydrophobic groups, wherein such hydrophobic groups comprise one or morenaturally occurring lipids such as cholesterol, and/or an optionallysubstituted, variably saturated or unsaturated C₆-C₂₀ alkyl and/or anoptionally substituted, variably saturated or unsaturated C₆-C₂₀ acyl.

Compound described herein may also comprise one or more isotopicsubstitutions. For example, H may be in any isotopic form, including ¹H,²H (D or deuterium), and ³H (T or tritium); C may be in any isotopicform, including ¹²C, ¹³C, ¹⁴C; O may be in any isotopic form, including¹⁶O and ¹⁸O; F may be in any isotopic form, including ¹⁸F and ¹⁹F; andthe like.

When describing the invention, which may include compounds andpharmaceutically acceptable salts thereof, pharmaceutical compositionscontaining such compounds and methods of using such compounds andcompositions, the following terms, if present, have the followingmeanings unless otherwise indicated. It should also be understood thatwhen described herein any of the moieties defined forth below may besubstituted with a variety of substituents, and that the respectivedefinitions are intended to include such substituted moieties withintheir scope as set out below. Unless otherwise stated, the term“substituted” is to be defined as set out below. It should be furtherunderstood that the terms “groups” and “radicals” can be consideredinterchangeable when used herein.

When a range of values is listed, it is intended to encompass each valueand sub-range within the range. For example, “C₁₋₆ alkyl” is intended toencompass, C₁, C₂, C₃, C₄, C₅, C₆, C₁₋₆, C₁₋₅, C₁₋₄, C₁₋₃, C₁₋₂, C₂₋₆,C₂₋₅, C₂₋₄, C₂₋₃, C₃₋₆, C₃₋₅, C₃₋₄, C₄₋₆, C₄₋₅, and C₅₋₆ alkyl.

In certain embodiments, the compounds disclosed herein comprise, forexample, at least one hydrophilic head-group and at least onehydrophobic tail-group, each bound to at least one cleavable group,thereby rendering such compounds amphiphilic. As used herein to describea compound or composition, the term “amphiphilic” means the ability todissolve in both polar (e.g., water) and non-polar (e.g., lipid)environments. For example, in certain embodiments, the compoundsdisclosed herein comprise at least one lipophilic tail-group (e.g.,cholesterol or a C₆-C₂₀ alkyl) and at least one hydrophilic head-group(e.g., imidazole), each bound to a cleavable group (e.g., disulfide).

It should be noted that the terms “head-group” and “tail-group” as useddescribe the compounds of the present invention, and in particularfunctional groups that comprise such compounds, are used for ease ofreference to describe the orientation of one or more functional groupsrelative to other functional groups. For example, in certain embodimentsa hydrophilic head-group (e.g., guanidinium) is bound (e.g., by one ormore of hydrogen-bonds, van der Waals' forces, ionic interactions andcovalent bonds) to a cleavable functional group (e.g., a disulfidegroup), which in turn is bound to a hydrophobic tail-group (e.g.,cholesterol).

As used herein, the term “alkyl” refers to both straight and branchedchain C₁-C₄₀ hydrocarbons (e.g., C₆-C₂₀ hydrocarbons), and include bothsaturated and unsaturated hydrocarbons. In certain embodiments, thealkyl may comprise one or more cyclic alkyls and/or one or moreheteroatoms such as oxygen, nitrogen, or sulfur and may optionally besubstituted with substituents (e.g., one or more of alkyl, halo,alkoxyl, hydroxy, amino, aryl, ether, ester or amide). In certainembodiments, a contemplated alkyl includes (9Z,12Z)-octadeca-9,12-dien.The use of designations such as, for example, “C₆-C₂₀” is intended torefer to an alkyl (e.g., straight or branched chain and inclusive ofalkenes and alkyls) having the recited range carbon atoms. In someembodiments, an alkyl group has 1 to 10 carbon atoms (“C₁₋₁₀ alkyl”). Insome embodiments, an alkyl group has 1 to 9 carbon atoms (“C₁₋₉ alkyl”).In some embodiments, an alkyl group has 1 to 8 carbon atoms (“C₁₋₈alkyl”). In some embodiments, an alkyl group has 1 to 7 carbon atoms(“C₁₋₇ alkyl”). In some embodiments, an alkyl group has 1 to 6 carbonatoms (“C₁₋₆ alkyl”). In some embodiments, an alkyl group has 1 to 5carbon atoms (“C₁₋₅ alkyl”). In some embodiments, an alkyl group has 1to 4 carbon atoms (“C₁₋₄ alkyl”). In some embodiments, an alkyl grouphas 1 to 3 carbon atoms (“C₁₋₃ alkyl”). In some embodiments, an alkylgroup has 1 to 2 carbon atoms (“C₁₋₂ alkyl”). In some embodiments, analkyl group has 1 carbon atom (“C₁ alkyl”). Examples of C₁₋₆ alkylgroups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl,pentyl, hexyl, and the like.

As used herein, “alkenyl” refers to a radical of a straight-chain orbranched hydrocarbon group having from 2 to 20 carbon atoms, one or morecarbon-carbon double bonds (e.g., 1, 2, 3, or 4 carbon-carbon doublebonds), and optionally one or more carbon-carbon triple bonds (e.g., 1,2, 3, or 4 carbon-carbon triple bonds) (“C_(2_20) alkenyl”). In certainembodiments, alkenyl does not contain any triple bonds. In someembodiments, an alkenyl group has 2 to 10 carbon atoms (“C₂₋₁₀alkenyl”). In some embodiments, an alkenyl group has 2 to 9 carbon atoms(“C₂₋₉ alkenyl”). In some embodiments, an alkenyl group has 2 to 8carbon atoms (“C₂₋₈ alkenyl”). In some embodiments, an alkenyl group has2 to 7 carbon atoms (“C₂₋₇ alkenyl”). In some embodiments, an alkenylgroup has 2 to 6 carbon atoms (“C₂₋₆ alkenyl”). In some embodiments, analkenyl group has 2 to 5 carbon atoms (“C₂₋₅ alkenyl”). In someembodiments, an alkenyl group has 2 to 4 carbon atoms (“C₂₋₄ alkenyl”).In some embodiments, an alkenyl group has 2 to 3 carbon atoms (“C₂₋₃alkenyl”). In some embodiments, an alkenyl group has 2 carbon atoms (“C₂alkenyl”). The one or more carbon-carbon double bonds can be internal(such as in 2-butenyl) or terminal (such as in 1-butenyl). Examples ofC₂₋₄ alkenyl groups include ethenyl (C₂), 1-propenyl (C₃), 2-propenyl(C₃), 1-butenyl (C₄), 2-butenyl (C₄), butadienyl (C₄), and the like.Examples of C₂₋₆ alkenyl groups include the aforementioned C₂₋₄ alkenylgroups as well as pentenyl (C₅), pentadienyl (C₅), hexenyl (C₆), and thelike. Additional examples of alkenyl include heptenyl (C₇), octenyl(C₈), octatrienyl (C₈), and the like.

As used herein, “alkynyl” refers to a radical of a straight-chain orbranched hydrocarbon group having from 2 to 20 carbon atoms, one or morecarbon-carbon triple bonds (e.g., 1, 2, 3, or 4 carbon-carbon triplebonds), and optionally one or more carbon-carbon double bonds (e.g., 1,2, 3, or 4 carbon-carbon double bonds) (“C₂₋₂₀ alkynyl”). In certainembodiments, alkynyl does not contain any double bonds. In someembodiments, an alkynyl group has 2 to 10 carbon atoms (“C₂₋₁₀alkynyl”). In some embodiments, an alkynyl group has 2 to 9 carbon atoms(“C₂₋₉ alkynyl”). In some embodiments, an alkynyl group has 2 to 8carbon atoms (“C₂₋₈ alkynyl”). In some embodiments, an alkynyl group has2 to 7 carbon atoms (“C₂₋₇ alkynyl”). In some embodiments, an alkynylgroup has 2 to 6 carbon atoms (“C₂₋₆ alkynyl”). In some embodiments, analkynyl group has 2 to 5 carbon atoms (“C₂₋₅ alkynyl”). In someembodiments, an alkynyl group has 2 to 4 carbon atoms (“C₂₋₄ alkynyl”).In some embodiments, an alkynyl group has 2 to 3 carbon atoms (“C₂₋₃alkynyl”). In some embodiments, an alkynyl group has 2 carbon atoms (“C₂alkynyl”). The one or more carbon-carbon triple bonds can be internal(such as in 2-butynyl) or terminal (such as in 1-butynyl). Examples ofC₂₋₄ alkynyl groups include, without limitation, ethynyl (C₂),1-propynyl (C₃), 2-propynyl (C₃), 1-butynyl (C₄), 2-butynyl (C₄), andthe like. Examples of C₂₋₆ alkenyl groups include the aforementionedC₂₋₄ alkynyl groups as well as pentynyl (C₅), hexynyl (C₆), and thelike. Additional examples of alkynyl include heptynyl (C₇), octynyl(C₈), and the like.

As used herein, “alkylene,” “alkenylene,” and “alkynylene,” refer to adivalent radical of an alkyl, alkenyl, and alkynyl group respectively.When a range or number of carbons is provided for a particular“alkylene,” “alkenylene,” or “alkynylene,” group, it is understood thatthe range or number refers to the range or number of carbons in thelinear carbon divalent chain. “Alkylene,” “alkenylene,” and“alkynylene,” groups may be substituted or unsubstituted with one ormore substituents as described herein.

As used herein, the term “aryl” refers to aromatic groups (e.g.,monocyclic, bicyclic and tricyclic structures) containing six to tencarbons in the ring portion. The aryl groups may be optionallysubstituted through available carbon atoms and in certain embodimentsmay include one or more heteroatoms such as oxygen, nitrogen or sulfur.In some embodiments, an aryl group has six ring carbon atoms (“C₆ aryl”;e.g., phenyl). In some embodiments, an aryl group has ten ring carbonatoms (“C₁₀ aryl”; e.g., naphthyl such as 1-naphthyl and 2-naphthyl).

As used herein, “heteroaryl” refers to a radical of a 5-10 memberedmonocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 10electrons shared in a cyclic array) having ring carbon atoms and 1-4ring heteroatoms provided in the aromatic ring system, wherein eachheteroatom is independently selected from nitrogen, oxygen and sulfur(“5-10 membered heteroaryl”). In heteroaryl groups that contain one ormore nitrogen atoms, the point of attachment can be a carbon or nitrogenatom, as valency permits. Heteroaryl bicyclic ring systems can includeone or more heteroatoms in one or both rings. “Heteroaryl” includes ringsystems wherein the heteroaryl ring, as defined above, is fused with oneor more carbocyclyl or heterocyclyl groups wherein the point ofattachment is on the heteroaryl ring, and in such instances, the numberof ring members continue to designate the number of ring members in theheteroaryl ring system. “Heteroaryl” also includes ring systems whereinthe heteroaryl ring, as defined above, is fused with one or more arylgroups wherein the point of attachment is either on the aryl orheteroaryl ring, and in such instances, the number of ring membersdesignates the number of ring members in the fused (aryl/heteroaryl)ring system. Bicyclic heteroaryl groups wherein one ring does notcontain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and thelike) the point of attachment can be on either ring, i.e., either thering bearing a heteroatom (e.g., 2-indolyl) or the ring that does notcontain a heteroatom (e.g., 5-indolyl).

The term “cycloalkyl” refers to a monovalent saturated cyclic, bicyclic,or bridged cyclic (e.g., adamantyl) hydrocarbon group of 3-12, 3-8, 4-8,or 4-6 carbons, referred to herein, e.g., as “C₄₋₈cycloalkyl,” derivedfrom a cycloalkane. Exemplary cycloalkyl groups include, but are notlimited to, cyclohexanes, cyclopentanes, cyclobutanes and cyclopropanes.

As used herein, “heterocyclyl” or “heterocyclic” refers to a radical ofa 3- to 10-membered non-aromatic ring system having ring carbon atomsand 1 to 4 ring heteroatoms, wherein each heteroatom is independentlyselected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon(“3-10 membered heterocyclyl”). In heterocyclyl groups that contain oneor more nitrogen atoms, the point of attachment can be a carbon ornitrogen atom, as valency permits. A heterocyclyl group can either bemonocyclic (“monocyclic heterocyclyl”) or a fused, bridged or spiro ringsystem such as a bicyclic system (“bicyclic heterocyclyl”), and can besaturated or can be partially unsaturated. Heterocyclyl bicyclic ringsystems can include one or more heteroatoms in one or both rings.“Heterocyclyl” also includes ring systems wherein the heterocyclyl ring,as defined above, is fused with one or more carbocyclyl groups whereinthe point of attachment is either on the carbocyclyl or heterocyclylring, or ring systems wherein the heterocyclyl ring, as defined above,is fused with one or more aryl or heteroaryl groups, wherein the pointof attachment is on the heterocyclyl ring, and in such instances, thenumber of ring members continue to designate the number of ring membersin the heterocyclyl ring system. The terms “heterocycle,”“heterocyclyl,” “heterocyclyl ring,” “heterocyclic group,” “heterocyclicmoiety,” and “heterocyclic radical,” may be used interchangeably.

As used herein, “cyano” refers to —CN.

The terms “halo” and “halogen” as used herein refer to an atom selectedfrom fluorine (fluoro, —F), chlorine (chloro, —Cl), bromine (bromo,—Br), and iodine (iodo, —I). In certain embodiments, the halo group iseither fluoro or chloro.

The term “alkoxy,” as used herein, refers to an alkyl group which isattached to another moiety via an oxygen atom (—O(alkyl)). Non-limitingexamples include e.g., methoxy, ethoxy, propoxy, and butoxy.

As used herein, “oxo” refers to —C═O.

In general, the term “substituted”, whether preceded by the term“optionally” or not, means that at least one hydrogen present on a group(e.g., a carbon or nitrogen atom) is replaced with a permissiblesubstituent, e.g., a substituent which upon substitution results in astable compound, e.g., a compound which does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, orother reaction. Unless otherwise indicated, a “substituted” group has asubstituent at one or more substitutable positions of the group, andwhen more than one position in any given structure is substituted, thesubstituent is either the same or different at each position.

As used herein, “pharmaceutically acceptable salt” refers to those saltswhich are, within the scope of sound medical judgment, suitable for usein contact with the tissues of humans and lower animals without unduetoxicity, irritation, allergic response and the like, and arecommensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, Berge et al.,describes pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically acceptablesalts of the compounds of this invention include those derived fromsuitable inorganic and organic acids and bases. Examples ofpharmaceutically acceptable, nontoxic acid addition salts are salts ofan amino group formed with inorganic acids such as hydrochloric acid,hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid orwith organic acids such as acetic acid, oxalic acid, maleic acid,tartaric acid, citric acid, succinic acid or malonic acid or by usingother methods used in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like. Pharmaceutically acceptable salts derived from appropriatebases include alkali metal, alkaline earth metal, ammonium andN⁺(C₁₋₄alkyl)₄ salts. Representative alkali or alkaline earth metalsalts include sodium, lithium, potassium, calcium, magnesium, and thelike. Further pharmaceutically acceptable salts include, whenappropriate, nontoxic ammonium, quaternary ammonium, and amine cationsformed using counterions such as halide, hydroxide, carboxylate,sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.

In typical embodiments, the present invention is intended to encompassthe compounds disclosed herein, and the pharmaceutically acceptablesalts, pharmaceutically acceptable esters, tautomeric forms, polymorphs,and prodrugs of such compounds. In some embodiments, the presentinvention includes a pharmaceutically acceptable addition salt, apharmaceutically acceptable ester, a solvate (e.g., hydrate) of anaddition salt, a tautomeric form, a polymorph, an enantiomer, a mixtureof enantiomers, a stereoisomer or mixture of stereoisomers (pure or as aracemic or non-racemic mixture) of a compound described herein.

Compounds described herein can comprise one or more asymmetric centers,and thus can exist in various isomeric forms, e.g., enantiomers and/ordiastereomers. For example, the compounds described herein can be in theform of an individual enantiomer, diastereomer or geometric isomer, orcan be in the form of a mixture of stereoisomers, including racemicmixtures and mixtures enriched in one or more stereoisomer. Isomers canbe isolated from mixtures by methods known to those skilled in the art,including chiral high pressure liquid chromatography (HPLC) and theformation and crystallization of chiral salts; or preferred isomers canbe prepared by asymmetric syntheses. See, for example, Jacques et al.,Enantiomers, Racemates and Resolutions (Wiley Interscience, New York,1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistryof Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, Tables ofResolving Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ.of Notre Dame Press, Notre Dame, IN 1972). The invention additionallyencompasses compounds described herein as individual isomerssubstantially free of other isomers, and alternatively, as mixtures ofvarious isomers.

In certain embodiments the compounds and the transfer vehicles of whichsuch compounds are a component (e.g., lipid nanoparticles) exhibit anenhanced (e.g., increased) ability to transfect one or more targetcells. Accordingly, also provided herein are methods of transfecting oneor more target cells. Such methods generally comprise the step ofcontacting the one or more target cells with the compounds and/orpharmaceutical compositions disclosed herein such that the one or moretarget cells are transfected with the circular RNA encapsulated therein.As used herein, the terms “transfect” or “transfection” refer to theintracellular introduction of one or more encapsulated materials (e.g.,nucleic acids and/or polynucleotides) into a cell, or preferably into atarget cell. The term “transfection efficiency” refers to the relativeamount of such encapsulated material (e.g., polynucleotides) up-takenby, introduced into and/or expressed by the target cell which is subjectto transfection. In some embodiments, transfection efficiency may beestimated by the amount of a reporter polynucleotide product produced bythe target cells following transfection. In some embodiments, a transfervehicle has high transfection efficiency. In some embodiments, atransfer vehicle has at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, or 90% transfection efficiency.

As used herein, the term “liposome” generally refers to a vesiclecomposed of lipids (e.g., amphiphilic lipids) arranged in one or morespherical bilayer or bilayers. In certain embodiments, the liposome is alipid nanoparticle (e.g., a lipid nanoparticle comprising one or more ofthe ionizable lipid compounds disclosed herein). Such liposomes may beunilamellar or multilamellar vesicles which have a membrane formed froma lipophilic material and an aqueous interior that contains theencapsulated circRNA to be delivered to one or more target cells,tissues and organs. In certain embodiments, the compositions describedherein comprise one or more lipid nanoparticles. Examples of suitablelipids (e.g., ionizable lipids) that may be used to form the liposomesand lipid nanoparticles contemplated include one or more of thecompounds disclosed herein (e.g., HGT4001, HGT4002, HGT4003, HGT4004and/or HGT4005). Such liposomes and lipid nanoparticles may alsocomprise additional ionizable lipids such as C12-200, DLin-KC2-DMA,and/or HGT5001, helper lipids, structural lipids, PEG-modified lipids,MC3, DLinDMA, DLinkC2DMA, cKK-E12, ICE, HGT5000, DODAC, DDAB, DMRIE,DOSPA, DOGS, DODAP, DODMA, DMDMA, DODAC, DLenDMA, DMRIE, CLinDMA,CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA,DLin-K-XTC2-DMA, HGT4003, and combinations thereof.

As used herein, the phrases “non-cationic lipid”, “non-cationic helperlipid”, and “helper lipid” are used interchangeably and refer to anyneutral, zwitterionic or anionic lipid.

As used herein, the phrase “anionic lipid” refers to any of a number oflipid species that carry a net negative charge at a selected pH, such asphysiological pH.

As used herein, the phrase “biodegradable lipid” or “degradable lipid”refers to any of a number of lipid species that are broken down in ahost environment on the order of minutes, hours, or days ideally makingthem less toxic and unlikely to accumulate in a host over time. Commonmodifications to lipids include ester bonds, and disulfide bonds amongothers to increase the biodegradability of a lipid.

As used herein, the phrase “biodegradable PEG lipid” or “degradable PEGlipid” refers to any of a number of lipid species where the PEGmolecules are cleaved from the lipid in a host environment on the orderof minutes, hours, or days ideally making them less immunogenic. Commonmodifications to PEG lipids include ester bonds, and disulfide bondsamong others to increase the biodegradability of a lipid.

In certain embodiments of the present invention, the transfer vehicles(e.g., lipid nanoparticles) are prepared to encapsulate one or morematerials or therapeutic agents (e.g., circRNA). The process ofincorporating a desired therapeutic agent (e.g., circRNA) into atransfer vehicle is referred to herein as or “loading” or“encapsulating” (Lasic, et al., FEBS Lett., 312: 255-258, 1992). Thetransfer vehicle-loaded or -encapsulated materials (e.g., circRNA) maybe completely or partially located in the interior space of the transfervehicle, within a bilayer membrane of the transfer vehicle, orassociated with the exterior surface of the transfer vehicle.

As used herein, the term “structural lipid” refers to sterols and alsoto lipids containing sterol moieties.

As defined herein, “sterols” are a subgroup of steroids consisting ofsteroid alcohols.

As used herein, the term “structural lipid” refers to sterols and alsoto lipids containing sterol moieties.

As used herein, the term “PEG” means any polyethylene glycol or otherpolyalkylene ether polymer.

As generally defined herein, a “PEG-OH lipid” (also referred to hereinas “hydroxy-PEGylated lipid”) is a PEGylated lipid having one or morehydroxyl (—OH) groups on the lipid.

As used herein, a “phospholipid” is a lipid that includes a phosphatemoiety and one or more carbon chains, such as unsaturated fatty acidchains.

All nucleotide sequences disclosed herein can represent an RNA sequenceor a corresponding DNA sequence. It is understood that deoxythymidine(dT or T) in a DNA is transcribed into a uridine (U) in an RNA. As such,“T” and “U” are used interchangeably herein in nucleotide sequences.

The recitations “sequence identity” or, for example, comprising a“sequence 50% identical to,” as used herein, refer to the extent thatsequences are identical on a nucleotide-by-nucleotide basis or an aminoacid-by-amino acid basis over a window of comparison. Thus, a“percentage of sequence identity” may be calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser,Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn,Gln, Cys and Met) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity. Included are nucleotides and polypeptides having at leastabout 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99% or 100% sequence identity to any of the reference sequencesdescribed herein, typically where the polypeptide variant maintains atleast one biological activity of the reference polypeptide.

2. Vectors, Precursor RNA, and Circular RNA

Also provided herein are circular RNAs, precursor RNAs that cancircularize into the circular RNAs, and vectors (e.g., DNA vectors) thatcan be transcribed into the precursor RNAs or the circular RNAs.

Two types of spacers have been designed for improving precursor RNAcircularization and/or gene expression from circular RNA. The first typeof spacer is external spacer, i.e., present in a precursor RNA butremoved upon circularization. While not wishing to be bound by theory,it is contemplated that an external spacer may improve ribozyme-mediatedcircularization by maintaining the structure of the ribozyme itself andpreventing other neighboring sequence elements from interfering with itsfolding and function. The second type of spacer is internal spacer,i.e., present in a precursor RNA and retained in a resulting circularRNA. While not wishing to be bound by theory, it is contemplated that aninternal spacer may improve ribozyme-mediated circularization bymaintaining the structure of the ribozyme itself and preventing otherneighboring sequence elements, particularly the neighboring IRES andcoding region, from interfering with its folding and function. It isalso contemplated that an internal spacer may improve protein expressionfrom the IRES by preventing neighboring sequence elements, particularlythe intron elements, from hybridizing with sequences within the IRES andinhibiting its ability to fold into its most preferred and activeconformation.

For driving protein expression, the circular RNA comprises an IRESoperably linked to a protein coding sequence. Exemplary IRES sequencesare provided in Table 17 below. In some embodiments, the circular RNAdisclosed herein comprises an IRES sequence at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an IRES sequencein Table 17. In some embodiments, the circular RNA disclosed hereincomprises an IRES sequence in Table 17. Modifications of IRES andaccessory sequences are disclosed herein to increase or reduce IRESactivities, for example, by truncating the 5′ and/or 3′ ends of theIRES, adding a spacer 5′ to the IRES, modifying the 6 nucleotides 5′ tothe translation initiation site (Kozak sequence), modification ofalternative translation initiation sites, and creating chimeric/hybridIRES sequences. In some embodiments, the IRES sequence in the circularRNA disclosed herein comprises one or more of these modificationsrelative to a native IRES (e.g., a native IRES disclosed in Table 17).

In certain aspects, provided herein are circular RNA polynucleotidescomprising a 3′ post splicing group I intron fragment, optionally afirst spacer, an Internal Ribosome Entry Site (IRES), an expressionsequence, optionally a second spacer, and a 5′ post splicing group Iintron fragment. In some embodiments, these regions are in that order.In some embodiments, the circular RNA is made by a method providedherein or from a vector provided herein.

In certain embodiments, transcription of a vector provided herein (e.g.,comprising a 5′ homology region, a 3′ group I intron fragment,optionally a first spacer, an Internal Ribosome Entry Site (IRES), anexpression sequence, optionally a second spacer, a 5′ group I intronfragment, and a 3′ homology region) results in the formation of aprecursor linear RNA polynucleotide capable of circularizing. In someembodiments, this precursor linear RNA polynucleotide circularizes whenincubated in the presence of guanosine nucleotide or nucleoside (e.g.,GTP) and divalent cation (e.g., Mg²⁺).

In some embodiments, the vectors and precursor RNA polynucleotidesprovided herein comprise a first (5′) duplex forming region and a second(3′) duplex forming region. In certain embodiments, the first and secondhomology regions may form perfect or imperfect duplexes. Thus, incertain embodiments at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% of the first and second duplex formingregions may be base paired with one another. In some embodiments, theduplex forming regions are predicted to have less than 50% (e.g., lessthan 45%, less than 40%, less than 35%, less than 30%, less than 25%)base pairing with unintended sequences in the RNA (e.g., non-duplexforming region sequences). In some embodiments, including such duplexforming regions on the ends of the precursor RNA strand, and adjacent orvery close to the group I intron fragment, bring the group I intronfragments in close proximity to each other, increasing splicingefficiency. In some embodiments, the duplex forming regions are 3 to 100nucleotides in length (e.g., 3-75 nucleotides in length, 3-50nucleotides in length, 20-50 nucleotides in length, 35-50 nucleotides inlength, 5-25 nucleotides in length, 9-19 nucleotides in length). In someembodiments, the duplex forming regions are about 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49 or 50 nucleotides in length. In some embodiments, theduplex forming regions have a length of about 9 to about 50 nucleotides.In one embodiment, the duplex forming regions have a length of about 9to about 19 nucleotides. In some embodiments, the duplex forming regionshave a length of about 20 to about 40 nucleotides. In certainembodiments, the duplex forming regions have a length of about 30nucleotides.

In certain embodiments, the vectors, precursor RNA and circular RNAprovided herein comprise a first (5′) and/or a second (3′) spacer. Insome embodiments, including a spacer between the 3′ group I intronfragment and the IRES may conserve secondary structures in those regionsby preventing them from interacting, thus increasing splicingefficiency. In some embodiments, the first (between 3′ group I intronfragment and IRES) and second (between the expression sequence and 5′group I intron fragment) spacers comprise additional base pairingregions that are predicted to base pair with each other and not to thefirst and second duplex forming regions. In some embodiments, suchspacer base pairing brings the group I intron fragments in closeproximity to each other, further increasing splicing efficiency.Additionally, in some embodiments, the combination of base pairingbetween the first and second duplex forming regions, and separately,base pairing between the first and second spacers, promotes theformation of a splicing bubble containing the group I intron fragmentsflanked by adjacent regions of base pairing. Typical spacers arecontiguous sequences with one or more of the following qualities: 1)predicted to avoid interfering with proximal structures, for example,the IRES, expression sequence, or intron; 2) is at least 7 nt long andno longer than 100 nt; 3) is located after and adjacent to the 3′ intronfragment and/or before and adjacent to the 5′ intron fragment; and 4)contains one or more of the following: a) an unstructured region atleast 5 nt long, b) a region of base pairing at least 5 nt long to adistal sequence, including another spacer, and c) a structured region atleast 7 nt long limited in scope to the sequence of the spacer. Spacersmay have several regions, including an unstructured region, a basepairing region, a hairpin/structured region, and combinations thereof.In an embodiment, the spacer has a structured region with high GCcontent. In an embodiment, a region within a spacer base pairs withanother region within the same spacer. In an embodiment, a region withina spacer base pairs with a region within another spacer. In anembodiment, a spacer comprises one or more hairpin structures. In anembodiment, a spacer comprises one or more hairpin structures with astem of 4 to 12 nucleotides and a loop of 2 to 10 nucleotides. In anembodiment, there is an additional spacer between the 3′ group I intronfragment and the IRES. In an embodiment, this additional spacer preventsthe structured regions of the IRES from interfering with the folding ofthe 3′ group I intron fragment or reduces the extent to which thisoccurs. In some embodiments, the 5′ spacer sequence is at least 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or 30 nucleotides inlength. In some embodiments, the 5′ spacer sequence is no more than 100,90, 80, 70, 60, 50, 45, 40, 35 or 30 nucleotides in length. In someembodiments the 5′ spacer sequence is between 5 and 50, 10 and 50, 20and 50, 20 and 40, and/or 25 and 35 nucleotides in length. In certainembodiments, the 5′ spacer sequence is 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotidesin length. In one embodiment, the 5′ spacer sequence is a polyAsequence. In another embodiment, the 5′ spacer sequence is a polyACsequence. In one embodiment, a spacer comprises about 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, or 100% polyAC content. In one embodiment,a spacer comprises about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or100% polypyrimidine (C/T or C/U) content.

In certain embodiments, a 3′ group I intron fragment is a contiguoussequence at least 75% identical (e.g., at least 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to a 3′ proximalfragment of a natural group I intron including the 3′ splice sitedinucleotide and optionally the adjacent exon sequence at least 1 nt inlength (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30 ntin length) and at most the length of the exon. Typically, a 5′ group Iintron fragment is a contiguous sequence at least 75% identical (e.g.,at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% identical) to a 5′ proximal fragment of a natural group I intronincluding the 5′ splice site dinucleotide and optionally the adjacentexon sequence at least 1 nt in length (e.g., at least 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 25 or 30 nt in length) and at most the length of theexon. As described by Umekage et al. (2012), external portions of the 3′group I intron fragment and 5′ group I intron fragment are removed incircularization, causing the circular RNA provided herein to compriseonly the portion of the 3′ group I intron fragment formed by theoptional exon sequence of at least 1 nt in length and 5′ group I intronfragment formed by the optional exon sequence of at least 1 nt inlength, if such sequences were present on the non-circularized precursorRNA. The part of the 3′ group I intron fragment that is retained by acircular RNA is referred to herein as the post splicing 3′ group Iintron fragment. The part of the 5′ group I intron fragment that isretained by a circular RNA is referred to herein as the post splicing 5′group I intron fragment.

In certain embodiments, the vectors, precursor RNA and circular RNAprovided herein comprise an internal ribosome entry site (IRES).Inclusion of an IRES permits the translation of one or more open readingframes from a circular RNA (e.g., open reading frames that form theexpression sequence). The IRES element attracts a eukaryotic ribosomaltranslation initiation complex and promotes translation initiation. See,e.g., Kaufman et al., Nuc. Acids Res. (1991) 19:4485-4490; Gurtu et al.,Biochem. Biophys. Res. Comm. (1996) 229:295-298; Rees et al.,BioTechniques (1996) 20: 102-110; Kobayashi et al., BioTechniques (1996)21:399-402; and Mosser et al., BioTechniques 1997 22 150-161).

A multitude of IRES sequences are available and include sequencesderived from a wide variety of viruses, such as from leader sequences ofpicornaviruses such as the encephalomyocarditis virus (EMCV) UTR (Janget al. J. Virol. (1989) 63: 1651-1660), the polio leader sequence, thehepatitis A virus leader, the hepatitis C virus IRES, human rhinovirustype 2 IRES (Dobrikova et al., Proc. Natl. Acad. Sci. (2003) 100(25):15125-15130), an IRES element from the foot and mouth disease virus(Ramesh et al., Nucl. Acid Res. (1996) 24:2697-2700), a giardiavirusIRES (Garlapati et al., J. Biol. Chem. (2004) 279(5):3389-3397), and thelike.

In some embodiments, the IRES is an IRES sequence of Taura syndromevirus, Triatoma virus, Theiler's encephalomyelitis virus, Simian Virus40, Solenopsis invicta virus 1, Rhopalosiphum padi virus,Reticuloendotheliosis virus, Human poliovirus 1, Plautia stali intestinevirus, Kashmir bee virus, Human rhinovirus 2, Homalodisca coagulatavirus-1, Human Immunodeficiency Virus type 1, Himetobi P virus,Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, Foot and mouthdisease virus, Human enterovirus 71, Equine rhinitis virus, Ectropisobliqua picorna-like virus, Encephalomyocarditis virus, Drosophila CVirus, Human coxsackievirus B3, Crucifer tobamovirus, Cricket paralysisvirus, Bovine viral diarrhea virus 1, Black Queen Cell Virus, Aphidlethal paralysis virus, Avian encephalomyelitis virus, Acute beeparalysis virus, Hibiscus chlorotic ringspot virus, Classical swinefever virus, Human FGF2, Human SFTPA1, Human AML1/RUNX1, Drosophilaantennapedia, Human AQP4, Human AT1R, Human BAG-1, Human BCL2, HumanBiP, Human c-IAP1, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEF1,Mouse HIF1 alpha, Human n.myc, Mouse Gtx, Human p27kip1, HumanPDGF2/c-sis, Human p53, Human Pim-1, Mouse Rbm3, Drosophila reaper,Canine Scamper, Drosophila Ubx, Human UNR, Mouse UtrA, Human VEGF-A,Human XIAP, Drosophila hairless, S. cerevisiae TFIID, S. cerevisiaeYAP1, tobacco etch virus, turnip crinkle virus, EMCV-A, EMCV-B, EMCV-Bf,EMCV-Cf, EMCV pEC9, Picobirnavirus, HCV QC64, Human Cosavirus E/D, HumanCosavirus F, Human Cosavirus JMY, Rhinovirus NAT001, HRV14, HRV89,HRVC-02, HRV-A21, Salivirus A SH1, Salivirus FHB, Salivirus NG-J1, HumanParechovirus 1, Crohivirus B, Yc-3, Rosavirus M-7, Shanbavirus A,Pasivirus A, Pasivirus A 2, Echovirus E14, Human Parechovirus 5, AichiVirus, Hepatitis A Virus HA16, Phopivirus, CVA10, Enterovirus C,Enterovirus D, Enterovirus J, Human Pegivirus 2, GBV-C GT110, GBV-CK1737, GBV-C Iowa, Pegivirus A 1220, Pasivirus A 3, Sapelovirus,Rosavirus B, Bakunsa Virus, Tremovirus A, Swine Pasivirus 1, PLV-CHN,Pasivirus A, Sicinivirus, Hepacivirus K, Hepacivirus A, BVDV1, BorderDisease Virus, BVDV2, CSFV-PK15C, SF573 Dicistrovirus, HubeiPicorna-like Virus, CRPV, Salivirus A BN5, Salivirus A BN2, Salivirus A02394, Salivirus A GUT, Salivirus A CH, Salivirus A SZ1, Salivirus FHB,CVB3, CVB1, Echovirus 7, CVB5, EVA71, CVA3, CVA12, EV24 or an aptamer toeIF4G.

In some embodiments, the polynucleotides herein comprise an expressionsequence. In some embodiments, the expression sequence encodes atherapeutic protein.

In some embodiments, the circular RNA encodes two or more polypeptides.In some embodiments, the circular RNA is a bicistronic RNA. Thesequences encoding the two or more polypeptides can be separated by aribosomal skipping element or a nucleotide sequence encoding a proteasecleavage site. In certain embodiments, the ribosomai skipping elementencodes thosea-asigna virus 2A peptide (T2A), porcine teschovirus-1 2 Apeptide (P2A), foot-and-mouth disease virus 2 A peptide (F2A), equinerhinitis A vims 2A peptide (E2A), cytoplasmic polyhedrosis vims 2Apeptide (BmCPV 2A), or flacherie vims of B. mori 2A peptide (BmIFV 2A).

In certain embodiments, the vectors provided herein comprise a 3′ UTR.In some embodiments, the 3′ UTR is from human beta globin, human alphaglobin Xenopus beta globin, Xenopus alpha globin, human prolactin, humanGAP-43, human eEFlal, human Tau, human TNFα, dengue virus, hantavirussmall mRNA, bunyavirus small mRNA, turnip yellow mosaic virus, hepatitisC virus, rubella virus, tobacco mosaic virus, human IL-8, human actin,human GAPDH, human tubulin, hibiscus chlorotic ringspot virus, woodchuckhepatitis virus post translationally regulated element, sindbis virus,turnip crinkle virus, tobacco etch virus, or Venezuelan equineencephalitis virus.

In some embodiments, the vectors provided herein comprise a 5′ UTR. Insome embodiments, the 5′ UTR is from human beta globin, Xenopus laevisbeta globin, human alpha globin, Xenopus laevis alpha globin, rubellavirus, tobacco mosaic virus, mouse Gtx, dengue virus, heat shock protein70 kDa protein 1A, tobacco alcohol dehydrogenase, tobacco etch virus,turnip crinkle virus, or the adenovirus tripartite leader.

In some embodiments, a vector provided herein comprises a polyA regionexternal of the 3′ and/or 5′ group I intron fragments. In someembodiments the polyA region is at least 15, 30, or 60 nucleotides long.In some embodiments, one or both polyA regions is 15-50 nucleotideslong. In some embodiments, one or both polyA regions is 20-25nucleotides long. The polyA sequence is removed upon circularization.Thus, an oligonucleotide hybridizing with the polyA sequence, such as adeoxythymine oligonucleotide (oligo(dT)) conjugated to a solid surface(e.g., a resin), can be used to separate circular RNA from its precursorRNA. Other sequences can also be disposed 5′ to the 3′ group I intronfragment or 3′ to the 5′ group I intron fragment and a complementarysequence can similarly be used for circular RNA purification.

In some embodiments, the DNA (e.g., vector), linear RNA (e.g., precursorRNA), and/or circular RNA polynucleotide provided herein is between 300and 10000, 400 and 9000, 500 and 8000, 600 and 7000, 700 and 6000, 800and 5000, 900 and 5000, 1000 and 5000, 1100 and 5000, 1200 and 5000,1300 and 5000, 1400 and 5000, and/or 1500 and 5000 nucleotides inlength. In some embodiments, the polynucleotide is at least 300 nt, 400nt, 500 nt, 600 nt, 700 nt, 800 nt, 900 nt, 1000 nt, 1100 nt, 1200 nt,1300 nt, 1400 nt, 1500 nt, 2000 nt, 2500 nt, 3000 nt, 3500 nt, 4000 nt,4500 nt, or 5000 nt in length. In some embodiments, the polynucleotideis no more than 3000 nt, 3500 nt, 4000 nt, 4500 nt, 5000 nt, 6000 nt,7000 nt, 8000 nt, 9000 nt, or 10000 nt in length. In some embodiments,the length of a DNA, linear RNA, and/or circular RNA polynucleotideprovided herein is about 300 nt, 400 nt, 500 nt, 600 nt, 700 nt, 800 nt,900 nt, 1000 nt, 1100 nt, 1200 nt, 1300 nt, 1400 nt, 1500 nt, 2000 nt,2500 nt, 3000 nt, 3500 nt, 4000 nt, 4500 nt, 5000 nt, 6000 nt, 7000 nt,8000 nt, 9000 nt, or 10000 nt.

In some embodiments, provided herein is a vector. In certainembodiments, the vector comprises, in the following order, a) a 5′homology region, b) a 3′ group I intron fragment, c) optionally, a firstspacer sequence, d) an IRES, e) an expression sequence, f) optionally, asecond spacer sequence, g) a 5′ group I intron fragment, and h) a 3′homology region. In some embodiments, the vector comprises atranscriptional promoter upstream of the 5′ homology region. In certainembodiments, the precursor RNA comprises, in the following order, a) apolyA sequence, b) an external spacer, c) a 3′ group I intron fragment,d) a duplex forming region, e) an internal spacer, f) an IRES, g) anexpression sequence, h) a stop codon cassette, i) optionally, aninternal spacer, j) a duplex forming region capable of forming a duplexwith the duplex forming region of d, k) a 5′ group I intron fragment, 1)an external spacer, and m) a polyA sequence.

In some embodiments, provided herein is a precursor RNA. In certainembodiments, the precursor RNA is a linear RNA produced by in vitrotranscription of a vector provided herein. In some embodiments, theprecursor RNA comprises, in the following order, a) a 5′ homologyregion, b) a 3′ group I intron fragment, c) optionally, a first spacersequence, d) an IRES, e) an expression sequence, f) optionally, a secondspacer sequence, g) a 5′ group I intron fragment, and h) a 3′ homologyregion. The precursor RNA can be unmodified, partially modified orcompletely modified.

In certain embodiments, provided herein is a circular RNA. In certainembodiments, the circular RNA is a circular RNA produced by a vectorprovided herein. In some embodiments, the circular RNA is circular RNAproduced by circularization of a precursor RNA provided herein. In someembodiments, the circular RNA comprises, in the following sequence, a) afirst spacer sequence, b) an IRES, c) an expression sequence, and d) asecond spacer sequence. In some embodiments, the circular RNA furthercomprises the portion of the 3′ group I intron fragment that is 3′ ofthe 3′ splice site. In some embodiments, the circular RNA furthercomprises the portion of the 5′ group I intron fragment that is 5′ ofthe 5′ splice site. In some embodiments, the circular RNA is at least500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000 or4500 nucleotides in size. The circular RNA can be unmodified, partiallymodified or completely modified.

In some embodiments, the circular RNA provided herein has higherfunctional stability than mRNA comprising the same expression sequence.In some embodiments, the circular RNA provided herein has higherfunctional stability than mRNA comprising the same expression sequence,5 moU modifications, an optimized UTR, a cap, and/or a polyA tail.

In some embodiments, the circular RNA polynucleotide provided herein hasa functional half-life of at least 5 hours, 10 hours, 15 hours, 20hours. 30 hours, 40 hours, 50 hours, 60 hours, 70 hours or 80 hours. Insome embodiments, the circular RNA polynucleotide provided herein has afunctional half-life of 5-80, 10-70, 15-60, and/or 20-50 hours. In someembodiments, the circular RNA polynucleotide provided herein has afunctional half-life greater than (e.g., at least 1.5-fold greater than,at least 2-fold greater than) that of an equivalent linear RNApolynucleotide encoding the same protein. In some embodiments,functional half-life can be assessed through the detection of functionalprotein synthesis.

In some embodiments, the circular RNA polynucleotide provided herein hasa half-life of at least 5 hours, 10 hours, 15 hours, 20 hours. 30 hours,40 hours, 50 hours, 60 hours, 70 hours or 80 hours. In some embodiments,the circular RNA polynucleotide provided herein has a half-life of 5-80,10-70, 15-60, and/or 20-50 hours. In some embodiments, the circular RNApolynucleotide provided herein has a half-life greater than (e.g., atleast 1.5-fold greater than, at least 2-fold greater than) that of anequivalent linear RNA polynucleotide encoding the same protein. In someembodiments, the circular RNA polynucleotide, or pharmaceuticalcomposition thereof, has a functional half-life in a human cell greaterthan or equal to that of a pre-determined threshold value. In someembodiments the functional half-life is determined by a functionalprotein assay. For example in some embodiments, the functional half-lifeis determined by an in vitro luciferase assay, wherein the activity ofGaussia luciferase (GLuc) is measured in the media of human cells (e.g.HepG2) expressing the circular RNA polynucleotide every 1, 2, 6, 12, or24 hours over 1, 2, 3, 4, 5, 6, 7, or 14 days. In other embodiments, thefunctional half-life is determined by an in vivo assay, wherein levelsof a protein encoded by the expression sequence of the circular RNApolynucleotide are measured in patient serum or tissue samples every 1,2, 6, 12, or 24 hours over 1, 2, 3, 4, 5, 6, 7, or 14 days. In someembodiments, the pre-determined threshold value is the functionalhalf-life of a reference linear RNA polynucleotide comprising the sameexpression sequence as the circular RNA polynucleotide.

In some embodiments, the circular RNA provided herein may have a highermagnitude of expression than equivalent linear mRNA, e.g., a highermagnitude of expression 24 hours after administration of RNA to cells.In some embodiments, the circular RNA provided herein has a highermagnitude of expression than mRNA comprising the same expressionsequence, 5 moU modifications, an optimized UTR, a cap, and/or a polyAtail.

In some embodiments, the circular RNA provided herein may be lessimmunogenic than an equivalent mRNA when exposed to an immune system ofan organism or a certain type of immune cell. In some embodiments, thecircular RNA provided herein is associated with modulated production ofcytokines when exposed to an immune system of an organism or a certaintype of immune cell. For example, in some embodiments, the circular RNAprovided herein is associated with reduced production of IFN-β1, RIG-I,IL-2, IL-6, IFNγ, and/or TNFβ when exposed to an immune system of anorganism or a certain type of immune cell as compared to mRNA comprisingthe same expression sequence. In some embodiments, the circular RNAprovided herein is associated with less IFN-β1, RIG-I, IL-2, IL-6, IFNγ,and/or TNFα transcript induction when exposed to an immune system of anorganism or a certain type of immune cell as compared to mRNA comprisingthe same expression sequence. In some embodiments, the circular RNAprovided herein is less immunogenic than mRNA comprising the sameexpression sequence. In some embodiments, the circular RNA providedherein is less immunogenic than mRNA comprising the same expressionsequence, 5 moU modifications, an optimized UTR, a cap, and/or a polyAtail.

In certain embodiments, the circular RNA provided herein can betransfected into a cell as is, or can be transfected in DNA vector formand transcribed in the cell. Transcription of circular RNA from atransfected DNA vector can be via added polymerases or polymerasesencoded by nucleic acids transfected into the cell, or preferably viaendogenous polymerases.

In certain embodiments, a circular RNA polynucleotide provided hereincomprises modified RNA nucleotides and/or modified nucleosides. In someembodiments, the modified nucleoside is m⁵C (5-methylcytidine). Inanother embodiment, the modified nucleoside is m⁵U (5-methyluridine). Inanother embodiment, the modified nucleoside is m⁶A (N⁶-methyladenosine).In another embodiment, the modified nucleoside is s²U (2-thiouridine).In another embodiment, the modified nucleoside is Ψ (pseudouridine). Inanother embodiment, the modified nucleoside is Um (2′-O-methyluridine).In other embodiments, the modified nucleoside is m¹A(1-methyladenosine); m²A (2-methyladenosine); Am (2′-O-methyladenosine);ms² m⁶A (2-methylthio-N⁶-methyladenosine); i⁶A(N⁶-isopentenyladenosine); ms²i6A (2-methylthio-N⁶isopentenyladenosine); io⁶A (N⁶-(cis-hydroxyisopentenyl)adenosine);ms²io⁶A (2-methylthio-N⁶-(cis-hydroxyisopentenyl)adenosine); g⁶A(N⁶-glycinylcarbamoyladenosine); t⁶A (N⁶-threonylcarbamoyladenosine);ms²t⁶A (2-methylthio-N⁶-threonyl carbamoyladenosine); m⁶t⁶A(N⁶-methyl-N⁶-threonylcarbamoyladenosine);hn⁶A(N⁶-hydroxynorvalylcarbamoyladenosine); ms²hn⁶A(2-methylthio-N⁶-hydroxynorvalyl carbamoyladenosine); Ar(p)(2′-O-ribosyladenosine (phosphate)); I (inosine); m¹I(1-methylinosine);m¹Im (1,2′-O-dimethylinosine); m³C (3-methylcytidine); Cm(2′-O-methylcytidine); s²C (2-thiocytidine); ac⁴C (N⁴-acetylcytidine);f⁵C (5-formylcytidine); m⁵Cm (5,2′-O-dimethylcytidine); ac⁴Cm(N⁴-acetyl-2′-O-methylcytidine); k²C (lysidine); m¹G(1-methylguanosine); m²G (N²-methylguanosine); m⁷G (7-methylguanosine);Gm (2′-O-methylguanosine); m² ₂G (N²,N²-dimethylguanosine); m²Gm(N²,2′-O-dimethylguanosine); m² ₂Gm (N²,N²,2′-O-trimethylguanosine);Gr(p) (2′-O-ribosylguanosine(phosphate)); yW (wybutosine); o₂yW(peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodifiedhydroxywybutosine); imG (wyosine); mimG (methylwyosine); Q (queuosine);oQ (epoxyqueuosine); galQ (galactosyl-queuosine); manQ(mannosyl-queuosine); preQ₀ (7-cyano-7-deazaguanosine); preQ₁(7-aminomethyl-7-deazaguanosine); G⁺ (archaeosine); D (dihydrouridine);m⁵Um (5,2′-O-dimethyluridine); s⁴U (4-thiouridine); m⁵s²U(5-methyl-2-thiouridine); s²Um (2-thio-2′-O-methyluridine); acp³U(3-(3-amino-3-carboxypropyl)uridine); ho⁵U (5-hydroxyuridine); mo⁵U(5-methoxyuridine); cmo⁵U (uridine 5-oxyacetic acid); mcmo⁵U (uridine5-oxyacetic acid methyl ester); chm⁵U(5-(carboxyhydroxymethyl)uridine)); mchm⁵U(5-(carboxyhydroxymethyl)uridine methyl ester); mcm⁵U(5-methoxycarbonylmethyluridine); mcm⁵Um(5-methoxycarbonylmethyl-2′-O-methyluridine); mcm⁵s²U(5-methoxycarbonylmethyl-2-thiouridine); nm⁵S²U(5-aminomethyl-2-thiouridine); mnm⁵U (5-methylaminomethyluridine);mnm⁵s²U (5-methylaminomethyl-2-thiouridine); mnm⁵se²U(5-methylaminomethyl-2-selenouridine); ncm⁵U (5-carbamoylmethyluridine);ncm⁵Um (5-carbamoylmethyl-2′-O-methyluridine); cmnm⁵U(5-carboxymethylaminomethyluridine); cmnm⁵Um(5-carboxymethylaminomethyl-2′-O-methyluridine); cmnm⁵s²U(5-carboxymethylaminomethyl-2-thiouridine); m⁶ ₂A(N⁶,N⁶-dimethyladenosine); Im (2′-O-methylinosine); m⁴C(N⁴-methylcytidine); m⁴Cm (N⁴,2′-O-dimethylcytidine); hm⁵C(5-hydroxymethylcytidine); m³U (3-methyluridine); cm⁵U(5-carboxymethyluridine); m⁶Am (N⁶,2′-O-dimethyladenosine); m⁶ ₂Am(N⁶,N⁶,O-2′-trimethyladenosine); M^(2,7)G (N²,7-dimethylguanosine);m^(2,2,7)G (N²,N²,7-trimethylguanosine); m³Um (3,2′-O-dimethyluridine);m⁵D (5-methyldihydrouridine); f⁵Cm (5-formyl-2′-O-methylcytidine); m¹Gm(1,2′-O-dimethylguanosine); m¹Am (1,2′-O-dimethyladenosine); τm⁵U(5-taurinomethyluridine); τm⁵s²U (5-taurinomethyl-2-thiouridine));imG-14 (4-demethylwyosine); imG2 (isowyosine); or ac⁶A(N⁶-acetyladenosine).

In some embodiments, the modified nucleoside may include a compoundselected from the group of: pyridin-4-one ribonucleoside, 5-aza-uridine,2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine,2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine,5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine,5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine,1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine,1-taurinomethyl-4-thio-uridine, 5-methyl-uridine,1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine,2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine,dihydropseudouridine, 2-thio-dihydrouridine,2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine,4-methoxy-pseudouridine, 4-m ethoxy-2-thio-pseudouridine,5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine,5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine,1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine,2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine,7-deaza-8-aza-adenine, 7-deaza-2-aminopurine,7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine,7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine,N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine,2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine,N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine,7-methyladenine, 2-methylthio-adenine, 2-methoxy-adenine, inosine,1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine,7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine,6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine,1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine,8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine. Inanother embodiment, the modifications are independently selected fromthe group consisting of 5-methylcytosine, pseudouridine and1-methylpseudouridine.

In some embodiments, the modified ribonucleosides include5-methylcytidine, 5-methoxyuridine, 1-methyl-pseudouridine,N6-methyladenosine, and/or pseudouridine. In some embodiments, suchmodified nucleosides provide additional stability and resistance toimmune activation.

In particular embodiments, polynucleotides may be codon-optimized. Acodon optimized sequence may be one in which codons in a polynucleotideencoding a polypeptide have been substituted in order to increase theexpression, stability and/or activity of the polypeptide. Factors thatinfluence codon optimization include, but are not limited to one or moreof: (i) variation of codon biases between two or more organisms or genesor synthetically constructed bias tables, (ii) variation in the degreeof codon bias within an organism, gene, or set of genes, (iii)systematic variation of codons including context, (iv) variation ofcodons according to their decoding tRNAs, (v) variation of codonsaccording to GC %, either overall or in one position of the triplet,(vi) variation in degree of similarity to a reference sequence forexample a naturally occurring sequence, (vii) variation in the codonfrequency cutoff, (viii) structural properties of mRNAs transcribed fromthe DNA sequence, (ix) prior knowledge about the function of the DNAsequences upon which design of the codon substitution set is to bebased, and/or (x) systematic variation of codon sets for each aminoacid. In some embodiments, a codon optimized polynucleotide may minimizeribozyme collisions and/or limit structural interference between theexpression sequence and the IRES.

In certain embodiments circular RNA provided herein is produced inside acell. In some embodiments, precursor RNA is transcribed using a DNAtemplate (e.g., in some embodiments, using a vector provided herein) inthe cytoplasm by a bacteriophage RNA polymerase, or in the nucleus byhost RNA polymerase II and then circularized.

In certain embodiments, the circular RNA provided herein is injectedinto an animal (e.g., a human), such that a polypeptide encoded by thecircular RNA molecule is expressed inside the animal.

3. Payload

In some embodiments, the expression sequence encodes a therapeuticprotein. In some embodiments, the therapeutic protein is selected fromthe proteins listed in the following table.

Target cell/ Payload Sequence organ Preferred delivery formulation CD19CAR Any of sequences 309-314 T cells

BCMA CAR MALPVTALLLPLALLL HAARPDIVLTQSPASLA VSLGERATINCRASESVSVIGAHLIHWYQQKPG QPPKLLIYLASNLETGV PARFSGSGSGTDFTLTIS SLQAEDAAIYYCLQSRIFPRTFGQGTKLEIKGST SGSGKPGSGEGSTKGQ VQLVQSGSELKKPGAS VKVSCKASGYTFTDYINWVRQAPGQGLEWMG WINTETREPAYAYDFR GRFVFSLDTSVSTAYLQ ISSLKAEDTAVYYCARDYSYAMDYWGQGTLV T cells

TVSSAAATTTPAPRPPT PAPTIASQPLSLRPEACR PAAGGAVHTRGLDFAC DIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLY IFKQPFMRPVQTTQEED GCSCRFPEEEEGGCELR VKFSRSADAPAYQQGQNQLYNELNLGRREEYD VLDKRRGRDPEMGGKP RRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT KDTYDALHMQALPPR MAGE- A4 TCR TCR alpha chain:KNQVEQSPQSLIILEGK NCTLQCNYTVSPFSNLR WYKQDTGRGPVSLTIM TFSENTKSNGRYTATLDADTKQSSLHITASQLSD SASYICVVNHSGGSYIP TFGRGTSLIVHPYIQKP DPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSK DSDVYITDKTVLDMRS MDFKSNSAVAWSNKS DFACANAFNNSIIPEDTFFPSPESS TCR beta chain: DVKVTQSSRYLVKRTG T cells

EKVFLECVQDMDHEN MFWYRQDPGLGLRLIY FSYDVKMKEKGDIPEG YSVSREKKERFSLILESASTNQTSMYLCASSFL MTSGDPYEQYFGPGTR LTVTEDLKNVFPPEVA VFEPSEAEISHTQKATLVCLATGFYPDHVELSW WVNGKEVHSGVSTDPQ PLKEQPALNDSRYCLSS RLRVSATFWQNPRNHFRCQVQFYGLSENDEWT QDRAKPVTQIVSAEAW GRAD NY- ESO TCR TCRalphaextracellular sequence MQEVTQIPAALSVPEGE NLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQ SSQREQTSGRLNASLDK SSGRSTLYIAASQPGDS ATYLCAVRPTSGGSYIPTFGRGTSLIVHPY TCRbeta cellular sequence MGVTQTPKFQVLKTGQ SMTLQCAQDMNHEYMSWYRQDPGMGLRLIHY SVGAGITDQGEVPNGY NVSRSTTEDFPLRLLSA T cells

APSQTSVYFCASSYVG NTGELFFGEGSRLTVL EPO APPRLICDSRVLERYLL KidneyEAKEAENITTGCAEHCS or LNENITVPDTKVNFYA bone WKRMEVGQQAVEVW marrowQGLALLSEAVLRGQAL LVNSSQPWEPLQLHVD KAVSGLRSLTTLLRALG AQKEAISPPDAASAAPLRTITADTFRKLFRVYSN FLRGKLKLYTGEACRT GDR PAH MSTAVLENPGLGRKLSDFGQETSYIEDNCNQN GAISLIFSLKEEVGALA KVLRLFEENDVNLTHIE SRPSRLKKDEYEFFTHLDKRSLPALTNIIKILRHD IGATVHELSRDKKKDT VPWFPRTIQELDRFANQ ILSYGAELDADHPGFKDPVYRARRKQFADIAYN YRHGQPIPRVEYMEEE KKTWGTVFKTLKSLYK THACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQF LQTCTGFRLRPVAGLLS SRDFLGGLAFRVFHCT QYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFA QFSQEIGLASLGAPDEY IEKLATIYWFTVEFGLC KQGDSIKAYGAGLLSFHepatic cells

GELQYCLSEDPKLLPLE LEKTAIQNYTVTEFQPL YYVAESFNDAKEKVRN FAATIPRPFSVRYDPYTQRIEVLDNTQQLKILAD SINSEIGILCSALQKIK CPS 1 LSVKAQTAHIVLEDGTKMKGYSFGHPSSVAGE VVFNTGLGGYPEAITDP AYKGQILTMANPIIGNG GAPDTTALDELGLSKYKDYNHWLATSLGQW LQEEKVPAIYGVDTRM LTKIIRDKGTMLGKEIF EGQPVDFVDPNKQNLIAEVSTKDVKVYGKGNP TKVVAVDCGIKNNVIR LLVKRGAEVHLVPWN HDFTKMEYDGILIAGGPGNPALAEPLIQNVRKIL ESDRKEPLFGISTGNLIT GLAAGAKTYKMSMAN RGQNQPVLNITNKQAFITAQNHGYALDNTLPAG WKPLFVNVNDQTNEGI MHESKPFFAVQFHPEV TPGPIDTEYLFDSFFSLIKKGKATTITSVLPKPAL Hepatic cells

VASRVEVSKVLILGSGG LSIGQAGEFDYSGSQAV KAMKEENVKTVLMNP NIASVQTNEVGLKQADTVYFLPITPQFVTEVIKA EQPDGLILGMGGQTAL NCGVELFKRGVLKEYG VKVLGTSVESIMATEDRQLFSDKLNEINEKIAPS FAVESIEDALKAADTIG YPVMIRSAYALGGLGS GICPNRETLMDLSTKAFAMTNQILVEKSVTGWK EIEYEVVRDADDNCVT VCNMENVDAMGVHTG DSVVVAPAQTLSNAEFQMLRRTSINVVRHLGIV GECNIQFALHPTSMEYC IIEVNARLSRSSALASK ATGYPLAFIAAKIALGIPLPEIKNVVSGKTSACFE PSLDYMVTKIPRWDLD RFHGTSSRIGSSMKSVG EVMAIGRTFEESFQKALRMCHPSIEGFTPRLPMN KEWPSNLDLRKELSEPS STRIYAIAKAIDDNMSL DEIEKLTYIDKWFLYKMRDILNMEKTLKGLNS ESMTEETLKRAKEIGFS DKQISKCLGLTEAQTRE LRLKKNIHPWVKQIDTLAAEYPSVTNYLYVTYN GQEHDVNFDDHGMMV LGCGPYHIGSSVEFDW CAVSSIRTLRQLGKKTVVVNCNPETVSTDFDEC DKLYFEELSLERILDIYH QEACGGCIISVGGQIPN NLAVPLYKNGVKIMGTSPLQIDRAEDRSIFSAVL DELKVAQAPWKAVNT LNEALEFAKSVDYPCLL RPSYVLSGSAMNVVFSEDEMKKFLEEATRVSQ EHPVVLTKFVEGAREV EMDAVGDKGRVISHAI SEHVEDAGVHSGDATLMLPTQTISQGAIEKVKD ATRKIAKAFAISGPFNV QFLVKGNDVLVIECNL RASRSFPFVSKTLGVDFIDVATKVMIGENVDEK HLPTLDHPIIPADYVAIK APMFSWPRLRDADPILR CEMASTGEVACFGEGIHTAFLKAMLSTGFKIPQ KGILIGIQQSFRPRFLGV AEQLHNEGFKLFATEA TSDWLNANNVPATVAWPSQEGQNPSLSSIRKLI RDGSIDLVINLPNNNTK FVHDNYVIRRTAVDSGI PLLTNFQVTKLFAEAVQKSRKVDSKSLFHYRQ YSAGKAA Cas9 MKRNYILGLDIGITSVG YGIIDYETRDVIDAGVRLFKEANVENNEGRRSK RGARRLKRRRRHRIQR VKKLLFDYNLLTDHSE LSGINPYEARVKGLSQKLSEEEFSAALLHLAKRR GVHNVNEVEEDTGNEL STKEQISRNSKALEEKY VAELQLERLKKDGEVRGSINRFKTSDYVKEAK QLLKVQKAYHQLDQSF IDTYIDLLETRRTYYEG PGEGSPFGWKDIKEWYEMLMGHCTYFPEELRS VKYAYNADLYNALND Im- mune cells

LNNLVITRDENEKLEYY EKFQIIENVFKQKKKPT LKQIAKEILVNEEDIKG YRVTSTGKPEFTNLKVYHDIKDITARKEIINAE LLDQIAKILTIYQSSEDI QEELTNLNSELTQEEIE QISNLKGYTGTHNLSLKAINLILDELWHTNDNQI AIFNRLKLVPKKVDLSQ QKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKY GLPNDIIIELAREKNSKD AQKMINEMQKRNRQT NERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLY SLEAIPLEDLLNNPFNY EVDHIIPRSVSFDNSFNN KVLVKQEENSKKGNRTPFQYLSSSDSKISYETFK KHILNLAKGKGRISKTK KEYLLEERDINRFSVQK DFINRNLVDTRYATRGLMNLLRSYFRVNNLDV KVKSINGGFTSFLRRK WKFKKERNKGYKHHA EDALIIANADFIFKEWKKLDKAKKVMENQMFE EKQAESMPEIETEQEYK EIFITPHQIKHIKDFKDY KYSHRVDKKPNRELINDTLYSTRKDDKGNTLI VNNLNGLYDKDNDKL KKLINKSPEKLLMYHH DPQTYQKLKLIMEQYGDEKNPLYKYYEETGNY LTKYSKKDNGPVIKKIK YYGNKLNAHLDITDDY PNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVK NLDVIKKENYYEVNSK CYEEAKKLKKISNQAEF IASFYNNDLIKINGELYRVIGVNNDLLNRIEVN MIDITYREYLENMNDK RPPRIIKTIASKTQSIKK YSTDILGNLYEVKSKKHPQIIKKG ADAM TS13 AAGGILHLELLVAVGP DVFQAHQEDTERYVLT NLNIGAELLRDPSLGAQFRVHLVKMVILTEPEG APNITANLTSSLLSVCG WSQTINPEDDTDPGHA DLVLYITRFDLELPDGNRQVRGVTQLGGACSPT WSCLITEDTGFDLGVTI AHEIGHSFGLEHDGAPG SGCGPSGHVMASDGAAPRAGLAWSPCSRRQLL SLLSAGRARCVWDPPR PQPGSAGHPPDAQPGL YYSANEQCRVAFGPKAVACTFAREHLDMCQAL SCHTDPLDQSSCSRLLV PLLDGTECGVEKWCSK GRCRSLVELTPIAAVHGRWSSWGPRSPCSRSCG GGVVTRRRQCNNPRPA Hepatic cells

FGGRACVGADLQAEM CNTQACEKTQLEFMSQ QCARTDGQPLRSSPGG ASFYHWGAAVPHSQGDALCRHMCRAIGESFIM KRGDSFLDGTRCMPSG PREDGTLSLCVSGSCRT FGCDGRMDSQQVWDRCQVCGGDNSTCSPRKG SFTAGRAREYVTFLTVT PNLTSVYIANHRPLFTH LAVRIGGRYVVAGKMSISPNTTYPSLLEDGRVE YRVALTEDRLPRLEEIRI WGPLQEDADIQVYRRY GEEYGNLTRPDITFTYFQPKPRQAWVWAAVRG PCSVSCGAGLRWVNYS CLDQARKELVETVQCQ GSQQPPAWPEACVLEPCPPYWAVGDFGPCSAS CGGGLRERPVRCVEAQ GSLLKTLPPARCRAGA QQPAVALETCNPQPCPARWEVSEPSSCTSAGG AGLALENETCVPGADG LEAPVTEGPGSVDEKLP APEPCVGMSCPPGWGHLDATSAGEKAPSPWGSI RTGAQAAHVWTPAAG SCSVSCGRGLMELRFLC MDSALRVPVQEELCGLASKPGSRREVCQAVPC PARWQYKLAACSVSCG RGVVRRILYCARAHGE DDGEEILLDTQCQGLPRPEPQEACSLEPCPPRWK VMSLGPCSASCGLGTA RRSVACVQLDQGQDVE VDEAACAALVRPEASVPCLIADCTYRWHVGTW MECSVSCGDGIQRRRD TCLGPQAQAPVPADFC QHLPKPVTVRGCWAGPCVGQGTPSLVPHEEAA APGRTTATPAGASLEW SQARGLLFSPAPQPRRL LPGPQENSVQSSACGRQHLEPTGTIDMRGPGQ ADCAVAIGRPLGEVVT LRVLESSLNCSAGDML LLWGRLTWRKMCRKLLDMTFSSKTNTLVVRQ RCGRPGGGVLLRYGSQ LAPETFYRECDMQLFG PWGEIVSPSLSPATSNAGGCRLFINVAPHARIAI HALATNMGAGTEGAN ASYILIRDTHSLRTTAFH GQQVLYWESESSQAEMEFSEGFLKAQASLRGQ YWTLQSWVPEMQDPQ SWKGKEGT FOXP3 MPNPRPGKPSAPSLALGPSPGASPSWRAAPKAS DLLGARGPGGTFQGRD LRGGAHASSSSLNPMPP SQLQLPTLPLVMVAPSGARLGPLPHLQALLDR PHFMHQLSTVDAHART PVLQVHPLESPAMISLT PPTTATGVFSLKARPGLPPGINVASLEWVSREPA LLCTFPNPSAPRKDSTL SAVPQSSYPLLANGVC KWPGCEKVFEEPEDFLKHCQADHLLDEKGRA QCLLQREMVQSLEQQL VLEKEKLSAMQAHLAG Im- mune cells

KMALTKASSVASSDKG SCCIVAAGSQGPVVPA WSGPREAPDSLFAVRR HLWGSHGNSTFPEFLHNMDYFKFHNMRPPFTY ATLIRWAILEAPEKQRT LNEIYHWFTRMFAFFR NHPATWKNAIRHNLSLHKCFVRVESEKGAVWT VDELEFRKKRSQRPSRC SNPTPGP IL-10 SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRV KTFFQMKDQLDNLLLK ESLLEDFKGYLGCQALS EMIQFYLEEVMPQAENQDPDIKAHVNSLGENL KTLRLRLRRCHRFLPCE NKSKAVEQVKNAFNKL QEKGIYKAMSEFDIFINYIEAYMTMKIRN Im- mune cells

IL-2 APTSSSTKKTQLQLEHL LLDLQMILNGINNYKNP KLTRMLTFKFYMPKKATELKHLQCLEEELKPLE EVLNLAQSKNFHLRPR DLISNINVIVLELKGSET TFMCEYADETATIVEFLNRWITFCQSIISTLT Im- mune cells

In some embodiments, the expression sequence encodes a therapeuticprotein. In some embodiments, the expression sequence encodes acytokine, e.g., IL-12p70, IL-15, IL-2, IL-18, IL-21, IFN-α, IFN-β,IL-10, TGF-beta, IL-4, or IL-35, or a functional fragment thereof. Insome embodiments, the expression sequence encodes an immune checkpointinhibitor. In some embodiments, the expression sequence encodes anagonist (e.g., a TNFR family member such as CD137L, OX40L, ICOSL, LIGHT,or CD70). In some embodiments, the expression sequence encodes achimeric antigen receptor. In some embodiments, the expression sequenceencodes an inhibitory receptor agonist (e.g., PDL1, PDL2, Galectin-9,VISTA, B7H4, or MHCII) or inhibitory receptor (e.g., PD1, CTLA4, TIGIT,LAG3, or TIM3). In some embodiments, the expression sequence encodes aninhibitory receptor antagonist. In some embodiments, the expressionsequence encodes one or more TCR chains (alpha and beta chains or gammaand delta chains). In some embodiments, the expression sequence encodesa secreted T cell or immune cell engager (e.g., a bispecific antibodysuch as BiTE, targeting, e.g., CD3, CD137, or CD28 and a tumor-expressedprotein e.g., CD19, CD20, or BCMA etc.). In some embodiments, theexpression sequence encodes a transcription factor (e.g., FOXP3, HELIOS,TOX1, or TOX2). In some embodiments, the expression sequence encodes animmunosuppressive enzyme (e.g., IDO or CD39/CD73). In some embodiments,the expression sequence encodes a GvHD (e.g., anti-HLA-A2 CAR-Tregs).

In some embodiments, a polynucleotide encodes a protein that is made upof subunits that are encoded by more than one gene. For example, theprotein may be a heterodimer, wherein each chain or subunit of theprotein is encoded by a separate gene. It is possible that more than onecircRNA molecule is delivered in the transfer vehicle and each circRNAencodes a separate subunit of the protein. Alternatively, a singlecircRNA may be engineered to encode more than one subunit. In certainembodiments, separate circRNA molecules encoding the individual subunitsmay be administered in separate transfer vehicles.

3.1 Cytokines

Descriptions and/or amino acid sequences of IL-2, IL-7, IL-10, IL-12,IL-15, IL-18, IL-27beta, IFNgamma, and/or TGFbeta1 are provided hereinand at the www.uniprot.org database at accession numbers: P60568 (IL-2),P29459 (IL-12A), P29460 (IL-12B), P13232 (IL-7), P22301 (IL-10), P40933(IL-15), Q14116 (IL-18), Q14213 (IL-27beta), P01579 (IFNgamma), and/orP01137 (TGFbeta1).

3.2 PD-1 and PD-L1 Antagonists

In some embodiments, a PD-1 inhibitor is pembrolizumab, pidilizumab, ornivolumab. In some embodiments, Nivolumab is described in WO2006/121168.In some embodiments, Pembrolizumab is described in WO2009/114335. Insome embodiments, Pidilizumab is described in WO2009/101611. Additionalanti-PD1 antibodies are described in U.S. Pat. No. 8,609,089, US2010028330, US 20120114649, WO2010/027827 and WO2011/066342.

In some embodiments, a PD-L1 inhibitor is atezolizumab, avelumab,durvalumab, BMS-936559, or CK-301.

Descriptions and/or amino acid sequences of heavy and light chains ofPD-1, and/or PD-L1 antibodies are provided herein and at thewww.drugbank.ca database at accession numbers: DB09037 (Pembrolizumab),DB09035 (Nivolumab), DB15383 (Pidilizumab), DB11595 (Atezolizumab),DB11945 (Avelumab), and DB11714 (Durvalumab).

3.3 T Cell Receptors

TCRs are described using the International Immunogenetics (IMGT) TCRnomenclature, and links to the IMGT public database of TCR sequences.Native alpha-beta heterodimeric TCRs have an alpha chain and a betachain. Broadly, each chain may comprise variable, joining and constantregions, and the beta chain also usually contains a short diversityregion between the variable and joining regions, but this diversityregion is often considered as part of the joining region. Each variableregion may comprise three CDRs (Complementarity Determining Regions)embedded in a framework sequence, one being the hypervariable regionnamed CDR3. There are several types of alpha chain variable (Vα) regionsand several types of beta chain variable (Vβ) regions distinguished bytheir framework, CDR1 and CDR2 sequences, and by a partly defined CDR3sequence. The Vα types are referred to in IMGT nomenclature by a uniqueTRAV number. Thus, “TRAV21” defines a TCR Vα region having uniqueframework and CDR1 and CDR2 sequences, and a CDR3 sequence which ispartly defined by an amino acid sequence which is preserved from TCR toTCR but which also includes an amino acid sequence which varies from TCRto TCR. In the same way, “TRBV5-1” defines a TCR Vβ region having uniqueframework and CDR1 and CDR2 sequences, but with only a partly definedCDR3 sequence.

The joining regions of the TCR are similarly defined by the unique IMGTTRAJ and TRBJ nomenclature, and the constant regions by the IMGT TRACand TRBC nomenclature.

The beta chain diversity region is referred to in IMGT nomenclature bythe abbreviation TRBD, and, as mentioned, the concatenated TRBD/TRBJregions are often considered together as the joining region.

The unique sequences defined by the IMGT nomenclature are widely knownand accessible to those working in the TCR field. For example, they canbe found in the IMGT public database. The “T cell Receptor Factsbook”,(2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8 alsodiscloses sequences defined by the IMGT nomenclature, but because of itspublication date and consequent time-lag, the information thereinsometimes needs to be confirmed by reference to the IMGT database.

Native TCRs exist in heterodimeric αβ or γδ forms. However, recombinantTCRs consisting of aa or ββ homodimers have previously been shown tobind to peptide MHC molecules. Therefore, the TCR of the invention maybe a heterodimeric αβ TCR or may be an aa or ββ homodimeric TCR.

For use in adoptive therapy, an αβ heterodimeric TCR may, for example,be transfected as full length chains having both cytoplasmic andtransmembrane domains. In certain embodiments TCRs of the invention mayhave an introduced disulfide bond between residues of the respectiveconstant domains, as described, for example, in WO 2006/000830.

TCRs of the invention, particularly alpha-beta heterodimeric TCRs, maycomprise an alpha chain TRAC constant domain sequence and/or a betachain TRBC1 or TRBC2 constant domain sequence. The alpha and beta chainconstant domain sequences may be modified by truncation or substitutionto delete the native disulfide bond between Cys4 of exon 2 of TRAC andCys2 of exon 2 of TRBC1 or TRBC2. The alpha and/or beta chain constantdomain sequence(s) may also be modified by substitution of cysteineresidues for Thr 48 of TRAC and Ser 57 of TRBC1 or TRBC2, the saidcysteines forming a disulfide bond between the alpha and beta constantdomains of the TCR.

Binding affinity (inversely proportional to the equilibrium constantK_(D)) and binding half-life (expressed as T½) can be determined by anyappropriate method. It will be appreciated that doubling the affinity ofa TCR results in halving the K_(D). T½ is calculated as ln 2 divided bythe off-rate (koff). So doubling of T½ results in a halving in koff.K_(D) and koff values for TCRs are usually measured for soluble forms ofthe TCR, i.e. those forms which are truncated to remove cytoplasmic andtransmembrane domain residues. Therefore, it is to be understood that agiven TCR has an improved binding affinity for, and/or a bindinghalf-life for the parental TCR if a soluble form of that TCR has thesaid characteristics. Preferably the binding affinity or bindinghalf-life of a given TCR is measured several times, for example 3 ormore times, using the same assay protocol, and an average of the resultsis taken.

Since the TCRs of the invention have utility in adoptive therapy, theinvention includes a non-naturally occurring and/or purified and/orengineered cell, especially a T-cell, presenting a TCR of the invention.There are a number of methods suitable for the transfection of T cellswith nucleic acid (such as DNA, cDNA or RNA) encoding the TCRs of theinvention (see for example Robbins et al., (2008) J Immunol. 180:6116-6131). T cells expressing the TCRs of the invention will besuitable for use in adoptive therapy-based treatment of cancers such asthose of the pancreas and liver. As will be known to those skilled inthe art, there are a number of suitable methods by which adoptivetherapy can be carried out (see for example Rosenberg et al., (2008) NatRev Cancer 8(4): 299-308).

As is well-known in the art, TCRs of the invention may be subject topost-translational modifications when expressed by transfected cells.Glycosylation is one such modification, which may comprise the covalentattachment of oligosaccharide moieties to defined amino acids in the TCRchain. For example, asparagine residues, or serine/threonine residuesare well-known locations for oligosaccharide attachment. Theglycosylation status of a particular protein depends on a number offactors, including protein sequence, protein conformation and theavailability of certain enzymes. Furthermore, glycosylation status (i.e.oligosaccharide type, covalent linkage and total number of attachments)can influence protein function. Therefore, when producing recombinantproteins, controlling glycosylation is often desirable. Glycosylation oftransfected TCRs may be controlled by mutations of the transfected gene(Kuball J et al. (2009), J Exp Med 206(2):463-475). Such mutations arealso encompassed in this invention.

A TCR may be specific for an antigen in the group MAGE-A1, MAGE-A2,MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10,MAGE-A11, MAGE-A12, MAGE-A13, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5,GAGE-6, GAGE-7, GAGE-8, BAGE-1, RAGE-1, LB33/MUM-1, PRAME, NAG, MAGE-Xp2(MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (AGE-B4), tyrosinase, brainglycogen phosphorylase, Melan-A, MAGE-C1, MAGE-C2, NY-ESO-1, LAGE-1,SSX-1, SSX-2(HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1, CT-7,alpha-actinin-4, Bcr-Abl fusion protein, Casp-8, beta-catenin, cdc27,cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusionprotein, LDLR-fucosyltransferaseAS fusion protein, HLA-A2, HLA-A11,hsp70-2, KIAA0205, Mart2, Mum-2, and 3, neo-PAP, myosin class I, OS-9,pml-RARa fusion protein, PTPRK, K-ras, N-ras, Triosephosphate isomeras,GnTV, Herv-K-mel, Lage-1, Mage-C2, NA-88, Lage-2, SP17, and TRP2-Int2,(MART-I), gp100 (Pmel 17), TRP-1, TRP-2, MAGE-1, MAGE-3, p15(58), CEA,NY-ESO (LAGE), SCP-1, Hom/Mel-40, p53, H-Ras, HER-2/neu, BCR-ABL,E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA,human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5,MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9,CA 72-4, CAM 17.1, NuMa, K-ras, .beta.-Catenin, CDK4, Mum-1, p16, TAGE,PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, α-fetoprotein (AFP),13HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242,CA-50, CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175,M344, MA-50, MG7-Ag, MOV18, NB\170K, NY-CO-1, RCAS 1, SDCCAG16, TA-90(Mac-2 binding protein\cyclophilin C-associated protein), TAAL6, TAG72,TLP, and TPS.

3.4 Transcription Factors

Regulatory T cells (Treg) are important in maintaining homeostasis,controlling the magnitude and duration of the inflammatory response, andin preventing autoimmune and allergic responses.

In general, Tregs are thought to be mainly involved in suppressingimmune responses, functioning in part as a “self-check” for the immunesystem to prevent excessive reactions. In particular, Tregs are involvedin maintaining tolerance to self-antigens, harmless agents such aspollen or food, and abrogating autoimmune disease.

Tregs are found throughout the body including, without limitation, thegut, skin, lung, and liver. Additionally, Treg cells may also be foundin certain compartments of the body that are not directly exposed to theexternal environment such as the spleen, lymph nodes, and even adiposetissue. Each of these Treg cell populations is known or suspected tohave one or more unique features and additional information may be foundin Lehtimaki and Lahesmaa, Regulatory T cells control immune responsesthrough their non-redundant tissue specific features, 2013, FRONTIERS INIMMUNOL., 4(294): 1-10, the disclosure of which is hereby incorporatedin its entirety.

Typically, Tregs are known to require TGF-β and IL-2 for properactivation and development. Tregs, expressing abundant amounts of theIL-2 receptor (IL-2R), are reliant on IL-2 produced by activated Tcells. Tregs are known to produce both IL-10 and TGF-β, both potentimmunosuppressive cytokines. Additionally, Tregs are known to inhibitthe ability of antigen presenting cells (APCs) to stimulate T cells. Oneproposed mechanism for APC inhibition is via CTLA-4, which is expressedby Foxp3+ Treg. It is thought that CTLA-4 may bind to B7 molecules onAPCs and either block these molecules or remove them by causinginternalization resulting in reduced availability of B7 and an inabilityto provide adequate co-stimulation for immune responses. Additionaldiscussion regarding the origin, differentiation and function of Tregmay be found in Dhamne et al., Peripheral and thymic Foxp3+ regulatory Tcells in search of origin, distinction, and function, 2013, Frontiers inImmunol., 4 (253): 1-11, the disclosure of which is hereby incorporatedin its entirety.

Descriptions and/or amino acid sequences of FOXP3, STAT5B, and/or HELIOSare provided herein and at the www.uniprot.org database at accessionnumbers: Q9BZS1 (FOXP3), P51692 (STAT5b), and/or Q9UKS7 (HELIOS).

Foxp3

In some embodiments, a transcription factor is the Forkhead box P3transcription factor (Foxp3). Foxp3 has been shown to be a key regulatorin the differentiation and activity of Treg. In fact, loss-of-functionmutations in the Foxp3 gene have been shown to lead to the lethal IPEXsyndrome (immune dysregulation, polyendocrinopathy, enteropathy,X-linked). Patients with IPEX suffer from severe autoimmune responses,persistent eczema, and colitis. Regulatory T (Treg) cells expressingFoxp3 play a key role in limiting inflammatory responses in theintestine (Josefowicz, S. Z. et al. Nature, 2012, 482, 395-U1510).

STAT

Members of the signal transducer and activator of transcription (STAT)protein family are intracellular transcription factors that mediate manyaspects of cellular immunity, proliferation, apoptosis anddifferentiation. They are primarily activated by membranereceptor-associated Janus kinases (JAK). Dysregulation of this pathwayis frequently observed in primary tumors and leads to increasedangiogenesis, enhanced survival of tumors and immunosuppression. Geneknockout studies have provided evidence that STAT proteins are involvedin the development and function of the immune system and play a role inmaintaining immune tolerance and tumor surveillance.

There are seven mammalian STAT family members that have been identified:STAT1, STAT2, STAT3, STAT4, STAT5 (including STAT5A and STAT5B), andSTATE.

Extracellular binding of cytokines or growth factors induce activationof receptor-associated Janus kinases, which phosphorylate a specifictyrosine residue within the STAT protein promoting dimerization viatheir SH2 domains. The phosphorylated dimer is then actively transportedto the nucleus via an importin α/β ternary complex. Originally, STATproteins were described as latent cytoplasmic transcription factors asphosphorylation was thought to be required for nuclear retention.However, unphosphorylated STAT proteins also shuttle between the cytosoland nucleus, and play a role in gene expression. Once STAT reaches thenucleus, it binds to a consensus DNA-recognition motif calledgamma-activated sites (GAS) in the promoter region of cytokine-induciblegenes and activates transcription. The STAT protein can bedephosphorylated by nuclear phosphatases, which leads to inactivation ofSTAT and subsequent transport out of the nucleus by a exportin-RanGTPcomplex.

In some embodiments, a STAT protein of the present disclosure may be aSTAT protein that comprises a modification that modulates its expressionlevel or activity. In some embodiments such modifications include, amongother things, mutations that effect STAT dimerization, STAT proteinbinding to signaling partners, STAT protein localization or STAT proteindegradation. In some embodiments, a STAT protein of the presentdisclosure is constitutively active. In some embodiments, a STAT proteinof the present disclosure is constitutively active due to constitutivedimerization. In some embodiments, a STAT protein of the presentdisclosure is constitutively active due to constitutive phosphorylationas described in Onishi, M. et al., Mol. Cell. Biol. July 1998 vol. 18no. 7 3871-3879 the entirety of which is herein incorporated byreference.

3.5 Chimeric Antigen Receptors

Chimeric antigen receptors (CARs or CAR-Ts) are genetically-engineeredreceptors. These engineered receptors may be inserted into and expressedby immune cells, including T cells via circular RNA as described herein.With a CAR, a single receptor may be programmed to both recognize aspecific antigen and, when bound to that antigen, activate the immunecell to attack and destroy the cell bearing that antigen. When theseantigens exist on tumor cells, an immune cell that expresses the CAR maytarget and kill the tumor cell. In some embodiments, the CAR encoded bythe polynucleotide comprises (i) an antigen-binding molecule thatspecifically binds to a target antigen, (ii) a hinge domain, atransmembrane domain, and an intracellular domain, and (iii) anactivating domain.

In some embodiments, an orientation of the CARs in accordance with thedisclosure comprises an antigen binding domain (such as an scFv) intandem with a costimulatory domain and an activating domain. Thecostimulatory domain may comprise one or more of an extracellularportion, a transmembrane portion, and an intracellular portion. In otherembodiments, multiple costimulatory domains may be utilized in tandem.

Antigen Binding Domain

CARs may be engineered to bind to an antigen (such as a cell-surfaceantigen) by incorporating an antigen binding molecule that interactswith that targeted antigen. In some embodiments, the antigen bindingmolecule is an antibody fragment thereof, e.g., one or more single chainantibody fragment (scFv). An scFv is a single chain antibody fragmenthaving the variable regions of the heavy and light chains of an antibodylinked together. See U.S. Pat. Nos. 7,741,465, and 6,319,494 as well asEshhar et al., Cancer Immunol Immunotherapy (1997) 45: 131-136. An scFvretains the parent antibody's ability to specifically interact withtarget antigen. scFvs are useful in chimeric antigen receptors becausethey may be engineered to be expressed as part of a single chain alongwith the other CAR components. Id. See also Krause et al., J. Exp. Med.,Volume 188, No. 4, 1998 (619-626); Finney et al., Journal of Immunology,1998, 161: 2791-2797. It will be appreciated that the antigen bindingmolecule is typically contained within the extracellular portion of theCAR such that it is capable of recognizing and binding to the antigen ofinterest. Bispecific and multispecific CARs are contemplated within thescope of the invention, with specificity to more than one target ofinterest.

In some embodiments, the antigen binding molecule comprises a singlechain, wherein the heavy chain variable region and the light chainvariable region are connected by a linker. In some embodiments, the VHis located at the N terminus of the linker and the VL is located at theC terminus of the linker. In other embodiments, the VL is located at theN terminus of the linker and the VH is located at the C terminus of thelinker. In some embodiments, the linker comprises at least about 5, atleast about 8, at least about 10, at least about 13, at least about 15,at least about 18, at least about 20, at least about 25, at least about30, at least about 35, at least about 40, at least about 45, at leastabout 50, at least about 60, at least about 70, at least about 80, atleast about 90, or at least about 100 amino acids.

In some embodiments, the antigen binding molecule comprises a nanobody.In some embodiments, the antigen binding molecule comprises a DARPin. Insome embodiments, the antigen binding molecule comprises an anticalin orother synthetic protein capable of specific binding to target protein.

In some embodiments, the CAR comprises an antigen binding domainspecific for an antigen selected from the group CD19, CD123, CD22, CD30,CD171, CS-1, C-type lectin-like molecule-1, CD33, epidermal growthfactor receptor variant III (EGFRvIII), ganglioside G2 (GD2),ganglioside GD3, TNF receptor family member B cell maturation (BCMA), Tnantigen ((Tn Ag) or (GaINAca-Ser/Thr)), prostate-specific membraneantigen (PSMA), Receptor tyrosine kinase-like orphan receptor 1 (ROR1),Fms-Like Tyrosine Kinase 3 (FLT3), Tumor-associated glycoprotein 72(TAG72), CD38, CD44v6, Carcinoembryonic antigen (CEA), Epithelial celladhesion molecule (EPCAM), B7H3 (CD276), KIT (CD117), Interleukin-13receptor subunit alpha-2, mesothelin, Interleukin 11 receptor alpha(IL-11Ra), prostate stem cell antigen (PSCA), Protease Serine 21,vascular endothelial growth factor receptor 2 (VEGFR2), Lewis(Y)antigen, CD24, Platelet-derived growth factor receptor beta(PDGFR-beta), Stage-specific embryonic antigen-4 (SSEA-4), CD20, Folatereceptor alpha, HER2, HER3, Mucin 1, cell surface associated (MUC1),epidermal growth factor receptor (EGFR), neural cell adhesion molecule(NCAM), Prostase, prostatic acid phosphatase (PAP), elongation factor 2mutated (ELF2M), Ephrin B2, fibroblast activation protein alpha (FAP),insulin-like growth factor 1 receptor (IGF-I receptor), carbonicanhydrase IX (CAIX), Proteasome (Prosome, Macropain) Subunit, Beta Type,9 (LMP2), glycoprotein 100 (gp100), oncogene fusion protein consistingof breakpoint cluster region (BCR) and Abelson murine leukemia viraloncogene homolog 1 (Abl) (bcr-abl), tyrosinase, ephrin type-A receptor 2(EphA2), Fucosyl GM1, sialyl Lewis adhesion molecule (sLe), gangliosideGM3, transglutaminase 5 (TGS5), high molecularweight-melanoma-associated antigen (HMWMAA), o-acetyl-GD2 ganglioside(OAcGD2), Folate receptor beta, tumor endothelial marker 1 (TEM1/CD248),tumor endothelial marker 7-related (TEM7R), claudin 6 (CLDN6), thyroidstimulating hormone receptor (TSHR), G protein-coupled receptor class Cgroup 5, member D (GPRC5D), chromosome X open reading frame 61(CXORF61), CD97, CD179a, anaplastic lymphoma kinase (ALK), Polysialicacid, placenta-specific 1 (PLAC1), hexasaccharide portion of globoHglycoceramide (GloboH), mammary gland differentiation antigen (NY-BR-1),uroplakin 2 (UPK2), Hepatitis A virus cellular receptor 1 (HAVCR1),adrenoceptor beta 3 (ADRB3), pannexin 3 (PANX3), G protein-coupledreceptor 20 (GPR20), lymphocyte antigen 6 complex, locus K 9 (LY6K),Olfactory receptor 51E2 (OR51E2), TCR Gamma Alternate Reading FrameProtein (TARP), Wilms tumor protein (WT1), Cancer/testis antigen 1(NY-ESO-1), Cancer/testis antigen 2 (LAGE-1a), MAGE family members(including MAGE-A1, MAGE-A3 and MAGE-A4), ETS translocation-variant gene6, located on chromosome 12p (ETV6-AML), sperm protein 17 (SPA17), XAntigen Family, Member 1A (XAGE1), angiopoietin-binding cell surfacereceptor 2 (Tie 2), melanoma cancer testis antigen-1 (MAD-CT-1),melanoma cancer testis antigen-2 (MAD-CT-2), Fos-related antigen 1,tumor protein p53 (p53), p53 mutant, prostein, surviving, telomerase,prostate carcinoma tumor antigen-1, melanoma antigen recognized by Tcells 1, Rat sarcoma (Ras) mutant, human Telomerase reversetranscriptase (hTERT), sarcoma translocation breakpoints, melanomainhibitor of apoptosis (ML-IAP), ERG (transmembrane protease, serine 2(TMPRSS2) ETS fusion gene), N-Acetyl glucosaminyl-transferase V (NA17),paired box protein Pax-3 (PAX3), Androgen receptor, Cyclin B1, v-mycavian myelocytomatosis viral oncogene neuroblastoma derived homolog(MYCN), Ras Homolog Family Member C (RhoC), Tyrosinase-related protein 2(TRP-2), Cytochrome P450 1B1 (CYP1B1), CCCTC-Binding Factor (Zinc FingerProtein)-Like, Squamous Cell Carcinoma Antigen Recognized By T Cells 3(SART3), Paired box protein Pax-5 (PAX5), proacrosin binding proteinsp32 (OY-TES1), lymphocyte-specific protein tyrosine kinase (LCK), Akinase anchor protein 4 (AKAP-4), synovial sarcoma, X breakpoint 2(SSX2), Receptor for Advanced Glycation Endproducts (RAGE-1), renalubiquitous 1 (RU1), renal ubiquitous 2 (RU2), legumain, human papillomavirus E6 (HPV E6), human papilloma virus E7 (HPV E7), intestinalcarboxyl esterase, heat shock protein 70-2 mutated (mut hsp70-2), CD79a,CD79b, CD72, Leukocyte-associated immunoglobulin-like receptor 1(LAIR1), Fc fragment of IgA receptor (FCAR or CD89), Leukocyteimmunoglobulin-like receptor subfamily A member 2 (LILRA2), CD300molecule-like family member f (CD300LF), C-type lectin domain family 12member A (CLEC12A), bone marrow stromal cell antigen 2 (BST2), EGF-likemodule-containing mucin-like hormone receptor-like 2 (EMR2), lymphocyteantigen 75 (LY75), Glypican-3 (GPC3), Fc receptor-like 5 (FCRL5), MUC16,5T4, 8H9, αvβθ integrin, αvβ6 integrin, alphafetoprotein (AFP), B7-H6,ca-125, CA9, CD44, CD44v7/8, CD52, E-cadherin, EMA (epithelial membraneantigen), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40(EGP-40), ErbB4, epithelial tumor antigen (ETA), folate binding protein(FBP), kinase insert domain receptor (KDR), k-light chain, L1 celladhesion molecule, MUC18, NKG2D, oncofetal antigen (h5T4),tumor/testis-antigen 1B, GAGE, GAGE-1, BAGE, SCP-1, CTZ9, SAGE, CAGE,CT10, MART-1, immunoglobulin lambda-like polypeptide 1 (IGLL1),Hepatitis B Surface Antigen Binding Protein (HBsAg), viral capsidantigen (VCA), early antigen (EA), EBV nuclear antigen (EBNA), HHV-6p41early antigen, HHV-6B U94 latent antigen, HHV-6B p98 late antigen,cytomegalovirus (CMV) antigen, large T antigen, small T antigen,adenovirus antigen, respiratory syncytial virus (RSV) antigen,haemagglutinin (HA), neuraminidase (NA), parainfluenza type 1 antigen,parainfluenza type 2 antigen, parainfluenza type 3 antigen,parainfluenza type 4 antigen, Human Metapneumovirus (HMPV) antigen,hepatitis C virus (HCV) core antigen, HIV p24 antigen, human T-celllympotrophic virus (HTLV-1) antigen, Merkel cell polyoma virus small Tantigen, Merkel cell polyoma virus large T antigen, Kaposisarcoma-associated herpesvirus (KSHV) lytic nuclear antigen and KSHVlatent nuclear antigen. In some embodiments, an antigen binding domaincomprises SEQ ID NO: 321 and/or 322.

Hinge/Spacer Domain

In some embodiments, a CAR of the instant disclosure comprises a hingeor spacer domain. In some embodiments, the hinge/spacer domain maycomprise a truncated hinge/spacer domain (THD) the THD domain is atruncated version of a complete hinge/spacer domain (“CHD”). In someembodiments, an extracellular domain is from or derived from (e.g.,comprises all or a fragment of) ErbB2, glycophorin A (GpA), CD2, CD3delta, CD3 epsilon, CD3 gamma, CD4, CD7, CD8a, CD8[T CD1 la (IT GAL),CD1 lb (IT GAM), CD1 lc (ITGAX), CD1 ld (IT GAD), CD18 (ITGB2), CD19(B4), CD27 (TNFRSF7), CD28, CD28T, CD29 (ITGB1), CD30 (TNFRSF8), CD40(TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA6),CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3),CD66e (CEACAM5), CD69 (CLEC2), CD79A (B-cell antigen receptorcomplex-associated alpha chain), CD79B (B-cell antigen receptorcomplex-associated beta chain), CD84 (SLAMF5), CD96 (Tactile), CD100(SEMA4D), CD103 (ITGAE), CD134 (0X40), CD137 (4-1BB), CD150 (SLAMF1),CD158A (KIR2DL1), CD158B1 (KIR2DL2), CD158B2 (KIR2DL3), CD158C(KIR3DP1), CD158D (KIRDL4), CD158F1 (KIR2DL5A), CD158F2 (KIR2DL5B),CD158K (KIR3DL2), CD160 (BY55), CD162 (SELPLG), CD226 (DNAM1), CD229(SLAMF3), CD244 (SLAMF4), CD247 (CD3-zeta), CD258 (LIGHT), CD268(BAFFR), CD270 (TNFSF14), CD272 (BTLA), CD276 (B7-H3), CD279 (PD-1),CD314 (NKG2D), CD319 (SLAMF7), CD335 (NK-p46), CD336 (NK-p44), CD337(NK-p30), CD352 (SLAMF6), CD353 (SLAMF8), CD355 (CRT AM), CD357(TNFRSF18), inducible T cell co-stimulator (ICOS), LFA-1 (CD1 la/CD18),NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL-2R beta, IL-2R gamma, IL-7Ralpha, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1/CBP, a CD83ligand, Fc gamma receptor, MHC class 1 molecule, MHC class 2 molecule, aTNF receptor protein, an immunoglobulin protein, a cytokine receptor, anintegrin, activating NK cell receptors, a Toll ligand receptor, andfragments or combinations thereof. A hinge or spacer domain may bederived either from a natural or from a synthetic source.

In some embodiments, a hinge or spacer domain is positioned between anantigen binding molecule (e.g., an scFv) and a transmembrane domain. Inthis orientation, the hinge/spacer domain provides distance between theantigen binding molecule and the surface of a cell membrane on which theCAR is expressed. In some embodiments, a hinge or spacer domain is fromor derived from an immunoglobulin. In some embodiments, a hinge orspacer domain is selected from the hinge/spacer regions of IgG1, IgG2,IgG3, IgG4, IgA, IgD, IgE, IgM, or a fragment thereof. In someembodiments, a hinge or spacer domain comprises, is from, or is derivedfrom the hinge/spacer region of CD8 alpha. In some embodiments, a hingeor spacer domain comprises, is from, or is derived from the hinge/spacerregion of CD28. In some embodiments, a hinge or spacer domain comprisesa fragment of the hinge/spacer region of CD8 alpha or a fragment of thehinge/spacer region of CD28, wherein the fragment is anything less thanthe whole hinge/spacer region. In some embodiments, the fragment of theCD8 alpha hinge/spacer region or the fragment of the CD28 hinge/spacerregion comprises an amino acid sequence that excludes at least 1, atleast 2, at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10, at least 11, at least 12, at least 13,at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19, or at least 20 amino acids at the N-terminus or C-Terminus, orboth, of the CD8 alpha hinge/spacer region, or of the CD28 hinge/spacerregion.

Transmembrane Domain

The CAR of the present disclosure may further comprise a transmembranedomain and/or an intracellular signaling domain. The transmembranedomain may be designed to be fused to the extracellular domain of theCAR. It may similarly be fused to the intracellular domain of the CAR.In some embodiments, the transmembrane domain that naturally isassociated with one of the domains in a CAR is used. In some instances,the transmembrane domain may be selected or modified (e.g., by an aminoacid substitution) to avoid binding of such domains to the transmembranedomains of the same or different surface membrane proteins to minimizeinteractions with other members of the receptor complex. Thetransmembrane domain may be derived either from a natural or from asynthetic source. Where the source is natural, the domain may be derivedfrom any membrane-bound or transmembrane protein.

Transmembrane regions may be derived from (i.e. comprise) a receptortyrosine kinase (e.g., ErbB2), glycophorin A (GpA), 4-1BB/CD137,activating NK cell receptors, an immunoglobulin protein, B7-H3, BAFFR,BFAME (SEAMF8), BTEA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19,CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3epsilon, CD3 gamma, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7,CD84, CD8alpha, CD8beta, CD96 (Tactile), CD1 la, CD1 lb, CD1 lc, CD1 Id,CDS, CEACAM1, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gammareceptor, GADS, GITR, HVEM (EIGHTR), IA4, ICAM-1, ICAM-1, Ig alpha(CD79a), IE-2R beta, IE-2R gamma, IE-7R alpha, inducible T cellcostimulator (ICOS), integrins, ITGA4, ITGA4, ITGA6, IT GAD, ITGAE,ITGAE, IT GAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, EAT, LFA-1, LFA-1, aligand that specifically binds with CD83, LIGHT, LIGHT, LTBR, Ly9(CD229), lymphocyte function-associated antigen-1 (LFA-1; CD1-1a/CD18),MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1),OX-40, PAG/Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162),Signaling Lymphocytic Activation Molecules (SLAM proteins), SLAM(SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Ly108),SLAMF7, SLP-76, TNF receptor proteins, TNFR2, TNFSF14, a Toll ligandreceptor, TRANCE/RANKL, VLA1, or VLA-6, or a fragment, truncation, or acombination thereof.

In some embodiments, suitable intracellular signaling domain include,but are not limited to, activating Macrophage/Myeloid cell receptorsCSFR1, MYD88, CD14, TIE2, TLR4, CR3, CD64, TREM2, DAP10, DAP12, CD169,DECTIN1, CD206, CD47, CD163, CD36, MARCO, TIM4, MERTK, F4/80, CD91,C1QR, LOX-1, CD68, SRA, BAI-1, ABCA7, CD36, CD31, Lactoferrin, or afragment, truncation, or combination thereof.

In some embodiments, a receptor tyrosine kinase may be derived from(e.g., comprise) Insulin receptor (InsR), Insulin-like growth factor Ireceptor (IGF1R), Insulin receptor-related receptor (IRR), plateletderived growth factor receptor alpha (PDGFRa), platelet derived growthfactor receptor beta (PDGFRfi). KIT proto-oncogene receptor tyrosinekinase (Kit), colony stimulating factor 1 receptor (CSFR), fms relatedtyrosine kinase 3 (FLT3), fms related tyrosine kinase 1 (VEGFR-1),kinase insert domain receptor (VEGFR-2), fms related tyrosine kinase 4(VEGFR-3), fibroblast growth factor receptor 1 (FGFR1), fibroblastgrowth factor receptor 2 (FGFR2), fibroblast growth factor receptor 3(FGFR3), fibroblast growth factor receptor 4 (FGFR4), protein tyrosinekinase 7 (CCK4), neurotrophic receptor tyrosine kinase 1 (trkA),neurotrophic receptor tyrosine kinase 2 (trkB), neurotrophic receptortyrosine kinase 3 (trkC), receptor tyrosine kinase like orphan receptor1 (ROR1), receptor tyrosine kinase like orphan receptor 2 (ROR2), muscleassociated receptor tyrosine kinase (MuSK), MET proto-oncogene, receptortyrosine kinase (MET), macrophage stimulating 1 receptor (Ron), AXLreceptor tyrosine kinase (Axl), TYRO3 protein tyrosine kinase (Tyro3),MER proto-oncogene, tyrosine kinase (Mer), tyrosine kinase withimmunoglobulin like and EGF like domains 1 (TIE1), TEK receptor tyrosinekinase (TIE2), EPH receptor A1 (EphA1), EPH receptor A2 (EphA2), (EPHreceptor A3) EphA3, EPH receptor A4 (EphA4), EPH receptor A5 (EphA5),EPH receptor A6 (EphA6), EPH receptor A7 (EphA7), EPH receptor A8(EphA8), EPH receptor A10 (EphA10), EPH receptor B1 (EphB1), EPHreceptor B2 (EphB2), EPH receptor B3 (EphB3), EPH receptor B4 (EphB4),EPH receptor B6 (EphB6), ret proto oncogene (Ret), receptor-liketyrosine kinase (RYK), discoidin domain receptor tyrosine kinase 1(DDR1), discoidin domain receptor tyrosine kinase 2 (DDR2), c-rosoncogene 1, receptor tyrosine kinase (ROS), apoptosis associatedtyrosine kinase (Lmr1), lemur tyrosine kinase 2 (Lmr2), lemur tyrosinekinase 3 (Lmr3), leukocyte receptor tyrosine kinase (LTK), ALK receptortyrosine kinase (ALK), or serine/threonine/tyrosine kinase 1 (STYK1).

Costimulatory Domain

In certain embodiments, the CAR comprises a costimulatory domain. Insome embodiments, the costimulatory domain comprises 4-1BB (CD137),CD28, or both, and/or an intracellular T cell signaling domain. In apreferred embodiment, the costimulatory domain is human CD28, human4-1BB, or both, and the intracellular T cell signaling domain is humanCD3 zeta (ζ). 4-1BB, CD28, CD3 zeta may comprise less than the whole4-1BB, CD28 or CD3 zeta, respectively. Chimeric antigen receptors mayincorporate costimulatory (signaling) domains to increase their potency.See U.S. Pat. Nos. 7,741,465, and 6,319,494, as well as Krause et al.and Finney et al. (supra), Song et al., Blood 119:696-706 (2012); Kaloset al., Sci Transl. Med. 3:95 (2011); Porter et al., N. Engl. J. Med.365:725-33 (2011), and Gross et al., Amur. Rev. Pharmacol. Toxicol.56:59-83 (2016).

In some embodiments, a costimulatory domain comprises the amino acidsequence of SEQ ID NO: 318 or 320.

Intracellular Signaling Domain

The intracellular (signaling) domain of the engineered T cells disclosedherein may provide signaling to an activating domain, which thenactivates at least one of the normal effector functions of the immunecell. Effector function of a T cell, for example, may be cytolyticactivity or helper activity including the secretion of cytokines.

In some embodiments, suitable intracellular signaling domain include(e.g., comprise), but are not limited to 4-1BB/CD137, activating NK cellreceptors, an Immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8),BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD 19a, CD2,CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3gamma, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8alpha,CD8beta, CD96 (Tactile), CD1 la, CD1 lb, CD1 lc, CD1 ld, CDS, CEACAM1,CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor,GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Ig alpha (CD79a), IL-2R beta,IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS),integrins, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2,ITGB7, ITGB1, KIRDS2, LAT, LFA-1, ligand that specifically binds withCD83, LIGHT, LTBR, Ly9 (CD229), Ly108, lymphocyte function-associatedantigen-1 (LFA-1; CD1-1a/CD18), MHC class 1 molecule, NKG2C, NKG2D,NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG/Cbp, programmed death-1(PD-1), PSGL1, SELPLG (CD162), Signaling Lymphocytic ActivationMolecules (SLAM proteins), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244;2B4), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor proteins, TNFR2,TNFSF14, a Toll ligand receptor, TRANCE/RANKL, VLA1, or VLA-6, or afragment, truncation, or a combination thereof.

CD3 is an element of the T cell receptor on native T cells, and has beenshown to be an important intracellular activating element in CARs. Insome embodiments, the CD3 is CD3 zeta. In some embodiments, theactivating domain comprises an amino acid sequence at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, at leastabout 99%, or about 100% identical to the polypeptide sequence of SEQ IDNO: 319.

3.6 Trispecific Antigen-Binding Proteins and Bispecific Antigen-BindingProteins

Disclosed herein are circular RNA polypeptides encoding trispecificantigen-binding proteins (TRITEs), bispecific antigen-binding proteins(BITEs), functional fragments thereof, and pharmaceutical compositionsthereof. Recombinant expression vectors useful for making circular RNAencoding trispecific antigen-binding proteins or bispecific antigenbinding proteins, and cells comprising the inventive circular RNA arealso provided herein. Also provided are methods of using the disclosedtrispecific antigen-binding proteins or the bispecific antigen-bindingproteins in the prevention and/or treatment of liver diseases,conditions and disorders. The trispecific antigen-binding proteins arecapable of specifically binding to a target antigen, e.g., a cancerantigen, as well as CD3, TCR, CD16A, or NKp46, and a liver retentiondomain or a half-life extension domain, such as a domain binding humanserum albumin (HSA). In some embodiments, the TRITE or BITE is createdwithin a patient's liver post-administration of a composition comprisingthe inventive circular RNA polypeptides to a patient in need thereof.

In one aspect, trispecific antigen-binding proteins comprise a domain(A) which specifically binds to CD3, TCR, CD16A, or NKp46, a domain (B)which specifically binds to a half-life extension molecule or a liverretention molecule, and a domain (C) which specifically binds to atarget antigen, e.g., a cancer cell antigen. The three domains intrispecific antigen-binding proteins may be arranged in any order. Thus,it is contemplated that the domain order of the trispecificantigen-binding proteins are in any of the following orders:(A)-(B)-(C), (A)-(C)-(B), (B)-(A)-(C), (B)-(C)-(A), (C)-(B)-(A), or(C)-(A)-(B).

In some embodiments, the trispecific antigen-binding proteins have adomain order of (A)-(B)-(C). In some embodiments, the trispecificantigen-binding proteins have a domain order of (A)-(C)-(B). In someembodiments, the trispecific antigen binding proteins have a domainorder of (B)-(A)-(C). In some embodiments, the trispecificantigen-binding proteins have a domain order of (B)-(C)-(A). In someembodiments, the trispecific antigen-binding proteins have a domainorder of (C)-(B)-(A). In some embodiments, the trispecificantigen-binding proteins have a domain order of (C)-(A)-(B).

In an embodiment, a bispecific antigen-binding protein comprises adomain (A) which specifically binds to CD3, TCR, CD16A, or NKp46, and adomain (B) which specifically binds to a target antigen. The two domainsin a bispecific antigen-binding protein are arranged in any order. Thus,it is contemplated that the domain order of the bispecificantigen-binding proteins may be: (A)-(B), or (B)-(A).

The trispecific antigen-binding proteins or bispecific antigen-bindingproteins described herein are designed to allow specific targeting ofcells expressing a target antigen by recruiting cytotoxic T cells or NKcells. This improves efficacy compared to ADCC (antibody dependentcell-mediated cytotoxicity), which uses full length antibodies directedto a sole antigen and is not capable of directly recruiting cytotoxic Tcells. In contrast, by engaging CD3 molecules expressed specifically onthese cells, the trispecific antigen-binding proteins or bispecificantigen-binding proteins can crosslink cytotoxic T cells or NK cellswith cells expressing a target antigen in a highly specific fashion,thereby directing the cytotoxic potential of the recruited T cell or NKcell towards the target cell. The trispecific antigen-binding proteinsor bispecific antigen-binding proteins described herein engage cytotoxicT cells via binding to the surface-expressed CD3 proteins, which formpart of the TCR, or CD16A or NKp46, which activates NK cells.Simultaneous binding of several trispecific antigen-binding protein orbispecific antigen-binding proteins to CD3 and to a target antigenexpressed on the surface of particular cells causes T cell activationand mediates the subsequent lysis of the particular target antigenexpressing cell. Thus, trispecific antigen-binding or bispecificantigen-binding proteins are contemplated to display strong, specificand efficient target cell killing. In some embodiments, the trispecificantigen-binding proteins or bispecific antigen-binding proteinsdescribed herein stimulate target cell killing by cytotoxic T cells toeliminate pathogenic cells (e.g., tumor cells, virally or bacteriallyinfected cells, autoreactive T cells, etc). In some embodiments, cellsare eliminated selectively, thereby reducing the potential for toxicside effects. In some embodiments anti-41bb or CD137 binding domains areused as the t cell engager.

Immune Cell Binding Domain

The specificity of the response of T cells is mediated by therecognition of antigen (displayed in context of a majorhistocompatibility complex, MHC) by the TCR. As part of the TCR, CD3 isa protein complex that includes a CD3γ (gamma) chain, a CD3δ (delta)chain, and two CD3ε (epsilon) chains which are present on the cellsurface. CD3 associates with the α (alpha) and β (beta) chains of theTCR as well as CD3ζ (zeta) altogether to comprise the complete TCR.Clustering of CD3 on T cells, such as by immobilized anti-CD3 antibodiesleads to T cell activation similar to the engagement of the T cellreceptor but independent of its clone-typical specificity.

In one aspect, the bispecific and trispecific proteins described hereincomprise a domain which specifically binds to CD3. In one aspect, thetrispecific proteins described herein comprise a domain whichspecifically binds to human CD3. In some embodiments, the trispecificproteins described herein comprise a domain which specifically binds toCD3γ. In some embodiments, the trispecific proteins described hereincomprise a domain which specifically binds to CD36. In some embodiments,the trispecific proteins described herein comprise a domain whichspecifically binds to CD3ε.

In further embodiments, the trispecific proteins described hereincomprise a domain which specifically binds to the TCR. In certaininstances, the trispecific proteins described herein comprise a domainwhich specifically binds the α chain of the TCR. In certain instances,the trispecific proteins described herein comprise a domain whichspecifically binds the β chain of the TCR.

In some embodiments, a trispecific antigen binding protein or bispecificantigen binding protein comprises a NKp46 specific binder. In someembodiments, a trispecific antigen binding protein or bispecific antigenbinding protein comprises a CD16A specific binder.

In some embodiments, the CD3, TCR, NKp46, or CD16A binding domain of theantigen-binding protein can be any domain that binds to CD3, TCR, NKp46,or CD16A including but not limited to domains from a monoclonalantibody, a polyclonal antibody, a recombinant antibody, a humanantibody, a humanized antibody. In some instances, it is beneficial forthe CD3, TCR, NKp46, or CD16A binding domain to be derived from the samespecies in which the trispecific antigen-binding protein will ultimatelybe used in. For example, for use in humans, it may be beneficial for theCD3, TCR, NKp46, or CD16A binding domain of the trispecificantigen-binding protein to comprise human or humanized residues from theantigen binding domain of an antibody or antibody fragment.

Thus, in one aspect, the antigen-binding domain comprises a humanized orhuman antibody or an antibody fragment, or a murine antibody or antibodyfragment. In one embodiment, the humanized or human anti-CD3, TCR,NKp46, or CD16A binding domain comprises one or more (e.g., all three)light chain complementary determining region 1 (LC CDR1), light chaincomplementary determining region 2 (LC CDR2), and light chaincomplementary determining region 3 (LC CDR3) of a humanized or humananti-CD3, TCR, NKp46, or CD16A binding domain described herein, and/orone or more (e.g., all three) heavy chain complementary determiningregion 1 (HC CDR1), heavy chain complementary determining region 2 (HCCDR2), and heavy chain complementary determining region 3 (HC CDR3) of ahumanized or human anti-CD3, TCR, NKp46, or CD16A binding domaindescribed herein, e.g., a humanized or human anti-CD3, TCR, NKp46, orCD16A binding domain comprising one or more, e.g., all three, LC CDRsand one or more, e.g., all three, HC CDRs.

In some embodiments, the humanized or human anti-CD3, TCR, NKp46, orCD16A binding domain comprises a humanized or human heavy chain variableregion specific to CD3, TCR, NKp46, or CD16A where the heavy chainvariable region specific to CD3, TCR, NKp46, or CD16A comprises human ornon-human heavy chain CDRs in a human heavy chain framework region.

In certain instances, the complementary determining regions of the heavychain and/or the light chain are derived from known anti-CD3 antibodies,such as, for example, muromonab-CD3 (OKT3), otelixizumab (TRX4),teplizumab (MGA031), visilizumab (Nuvion), SP34, TR-66 or X35-3, VIT3,BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2,TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6,T3/RW2-8C8, T3/RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1 and WT-31.

In some embodiments, an anti-NKp46 binding domain comprises an antibodyor fragment thereof described in U.S. patent application Ser. No.16/451,051. In some embodiments, an anti-NKp46 binding domain comprisesthe antibodies BAB281, 9E2, 195314 or a fragment thereof.

In one embodiment, the anti-CD3, TCR, NKp46, or CD16A binding domain isa single chain variable fragment (scFv) comprising a light chain and aheavy chain of an amino acid sequence provided herein. In an embodiment,the anti-CD3, TCR, NKp46, or CD16A binding domain comprises: a lightchain variable region comprising an amino acid sequence having at leastone, two or three modifications (e.g., substitutions) but not more than30, 20 or 10 modifications (e.g., substitutions) of an amino acidsequence of a light chain variable region provided herein, or a sequencewith 95-99% identity with an amino acid sequence provided herein; and/ora heavy chain variable region comprising an amino acid sequence havingat least one, two or three modifications (e.g., substitutions) but notmore than 30, 20 or 10 modifications (e.g., substitutions) of an aminoacid sequence of a heavy chain variable region provided herein, or asequence with 95-99% identity to an amino acid sequence provided herein.In one embodiment, the humanized or human anti-CD3 binding domain is ascFv, and a light chain variable region comprising an amino acidsequence described herein, is attached to a heavy chain variable regioncomprising an amino acid sequence described herein, via a scFv linker.The light chain variable region and heavy chain variable region of ascFv can be, e.g., in any of the following orientations: light chainvariable region-scFv linker-heavy chain variable region or heavy chainvariable region-scFv linker-light chain variable region.

In some embodiments, CD3, TCR, NKp46, or CD16A binding domain oftrispecific antigen-binding protein has an affinity to CD3, TCR, NKp46,or CD16A on CD3, TCR, NKp46, or CD16A expressing cells with a KD of 1000nM or less, 500 nM or less, 200 nM or less, 100 nM or less, 80 nM orless, 50 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 1 nM orless, or 0.5 nM or less. In some embodiments, the CD3 binding domain ofMSLN trispecific antigen-binding protein has an affinity to CD3ε, γ, orδ with a KD of 1000 nM or less, 500 nM or less, 200 nM or less, 100 nMor less, 80 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, 5nM or less, 1 nM or less, or 0.5 nM or less. In further embodiments,CD3, TCR, NKp46, or CD16A binding domain of trispecific antigen-bindingprotein has low affinity to CD3, TCR, NKp46, or CD16A, i.e., about 100nM or greater.

The affinity to bind to CD3, TCR, NKp46, or CD16A can be determined, forexample, by the ability of the trispecific antigen-binding proteinitself or its CD3, TCR, NKp46, or CD16A binding domain to bind to CD3,TCR, NKp46, or CD16A coated on an assay plate; displayed on a microbialcell surface; in solution; etc. The binding activity of the trispecificantigen-binding protein itself or its CD3, TCR, NKp46, or CD16A bindingdomain of the present disclosure to CD3, TCR, NKp46, or CD16A can beassayed by immobilizing the ligand (e.g., CD3, TCR, NKp46, or CD16A) orthe trispecific antigen-binding protein itself or its CD3, TCR, NKp46,or CD16A binding domain, to a bead, substrate, cell, etc. Agents can beadded in an appropriate buffer and the binding partners incubated for aperiod of time at a given temperature. After washes to remove unboundmaterial, the bound protein can be released with, for example, SDS,buffers with a high pH, and the like and analyzed, for example, bySurface Plasmon Resonance (SPR).

In some embodiments, a bispecific antigen binding protein or bispecificantigen binding protein comprises a TCR binding domain. In someembodiments, a TCR binding domain is a viral antigen or a fragmentthereof. In some embodiments, a viral antigen is from the families:Retroviridae (e.g., human immunodeficiency viruses, such as HIV-1 (alsoreferred to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III; and otherisolates, such as HIV-LP; Picornaviridae (e.g., polio viruses, hepatitisA virus; enteroviruses, human Coxsackie viruses, rhinoviruses,echoviruses); Calciviridae (e.g., strains that cause gastroenteritis);Togaviridae (e.g., equine encephalitis viruses, rubella viruses);Flaviviridae (e.g., dengue viruses, encephalitis viruses, yellow feverviruses); Coronaviridae (e.g., coronaviruses); Rhabdoviridae (e.g.,vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g., Ebolaviruses); Paramyxoviridae (e.g., parainfluenza viruses, mumps virus,measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g.,influenza viruses); Bunyaviridae (e.g., Hantaan viruses, bunya viruses,phleboviruses and Nairo viruses); Arenaviridae (hemorrhagic feverviruses); Reoviridae (e.g., reoviruses, orbiviruses and rotaviruses);Bornaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae(parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses);Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus(HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpesvirus; Poxviridae (variola viruses, vaccinia viruses, pox viruses); andIridoviridae (e.g., African swine fever virus); and unclassified viruses(e.g., the agent of delta hepatitis (thought to be a defective satelliteof hepatitis B virus), Hepatitis C; Norwalk and related viruses, andastroviruses).

Linkers

In the trispecific proteins described herein, the domains are linked byinternal linkers L1 and L2, where L1 links the first and second domainof the trispecific proteins and L2 links the second and third domains ofthe trispecific proteins. In some embodiments, linkers L1 and L2 have anoptimized length and/or amino acid composition. In some embodiments,linkers L1 and L2 are the same length and amino acid composition. Inother embodiments, L1 and L2 are different. In certain embodiments,internal linkers L1 and/or L2 consist of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11 or 12 amino acid residues. Thus, in certain instances, theinternal linkers consist of about 12 or less amino acid residues. In thecase of 0 amino acid residues, the internal linker is a peptide bond. Incertain embodiments, internal linkers L1 and/or L2 consist of 15, 20 or25 amino acid residues. In some embodiments, these internal linkersconsist of about 3 to about 15, for example 8, 9 or 10 contiguous aminoacid residues. Regarding the amino acid composition of the internallinkers L1 and L2, peptides are selected with properties that conferflexibility to the trispecific proteins, do not interfere with thebinding domains as well as resist cleavage from proteases. For example,glycine and serine residues generally provide protease resistance.Examples of internal linkers suitable for linking the domains in thetrispecific proteins include but are not limited to (GS)n, (GGS)n,(GGGS)n, (GGSG)n, (GGSGG)n, (GGGGS)n, (GGGGG)n, or (GGG)n, wherein n is1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, internal linker L1and/or L2 is (GGGGS)4 or (GGGGS)₃.

Half-Life Extension Domain

Contemplated herein are domains which extend the half-life of anantigen-binding domain. Such domains are contemplated to include but arenot limited to Albumin binding domains, Fc domains, small molecules, andother half-life extension domains known in the art.

Human albumin (ALB) is the most abundant protein in plasma, present atabout 50 mg/ml and has a half-life of around 20 days in humans. ALBserves to maintain plasma pH, contributes to colloidal blood pressure,functions as carrier of many metabolites and fatty acids, and serves asa major drug transport protein in plasma.

Noncovalent association with albumin extends the elimination half-timeof short lived proteins.

In one aspect, the trispecific proteins described herein comprise ahalf-life extension domain, for example a domain which specificallybinds to ALB. In some embodiments, the ALB binding domain of atrispecific antigen-binding protein can be any domain that binds to ALBincluding but not limited to domains from a monoclonal antibody, apolyclonal antibody, a recombinant antibody, a human antibody, ahumanized antibody. In some embodiments, the ALB binding domain is asingle chain variable fragments (scFv), single-domain antibody such as aheavy chain variable domain (VH), a light chain variable domain (VL) anda variable domain (VHH) of camelid derived single domain antibody,peptide, ligand or small molecule entity specific for HSA. In certainembodiments, the ALB binding domain is a single-domain antibody. Inother embodiments, the HSA binding domain is a peptide. In furtherembodiments, the HSA binding domain is a small molecule. It iscontemplated that the HSA binding domain of MSLN trispecificantigen-binding protein is fairly small and no more than 25 kD, no morethan 20 kD, no more than 15 kD, or no more than 10 kD in someembodiments. In certain instances, the ALB binding is 5 kD or less if itis a peptide or small molecule entity.

The half-life extension domain of a trispecific antigen-binding proteinprovides for altered pharmacodynamics and pharmacokinetics of thetrispecific antigen-binding protein itself. As above, the half-lifeextension domain extends the elimination half-time. The half-lifeextension domain also alters pharmacodynamic properties includingalteration of tissue distribution, penetration, and diffusion of thetrispecific antigen-binding protein. In some embodiments, the half-lifeextension domain provides for improved tissue (including tumor)targeting, tissue distribution, tissue penetration, diffusion within thetissue, and enhanced efficacy as compared with a protein without ahalf-life extension domain. In one embodiment, therapeutic methodseffectively and efficiently utilize a reduced amount of the trispecificantigen-binding protein, resulting in reduced side effects, such asreduced non-tumor cell cytotoxicity.

Further, the binding affinity of the half-life extension domain can beselected so as to target a specific elimination half-time in aparticular trispecific antigen-binding protein. Thus, in someembodiments, the half-life extension domain has a high binding affinity.In other embodiments, the half-life extension domain has a mediumbinding affinity. In yet other embodiments, the half-life extensiondomain has a low or marginal binding affinity. Exemplary bindingaffinities include KD concentrations at 10 nM or less (high), between 10nM and 100 nM (medium), and greater than 100 nM (low). As above, bindingaffinities to ALB are determined by known methods such as SurfacePlasmon Resonance (SPR).

Liver Retention Domain

Contemplated herein are domains which allows for and promotes a higherretention of the trispecific antigen-binding protein within liver. Theliver retention domain of the trispecific antigen-binding protein isdirected to targeting a liver cell moiety. In an embodiment, a livercell includes but is not limited to a hepatocyte, hepatic stellate cell,sinusoidal endothelial cell.

In an embodiment, a liver cell contains a receptor that binds to a livertargeting moiety. In an embodiment, the liver targeting moiety includes,but is not limited to lactose, cyanuric chloride, cellobiose, polylsine,polyarginine, Mannose-6-phosphate, PDGF, human serum albumin,galactoside, galactosamine, linoleic acid, Apoliopoprotein A-1, AcetylCKNEKKNIERNNKLKQPP-amide, glycyrrhizin, lactobionic acid, Mannose-BSA,BSA, poly-ACO-HAS, KLGR peptide, hyaluronic acid, IFN-alpha, cRGDpeptide, 6-phosphate-HSA, retinol, lactobiotin, galactoside, pullulan,soybean steryglucoside, asialoorosomucoid, glycyrrhetinicacid/glycyrrhizin, linoleic acid, AMD3100, cleavable hyaluronicacid-glycyrrhetinic acid, Hepatitis B virus pre-S1 derived lipoprotein,Apo-A1, or LDL. In an embodiment, the liver cell receptor includes butis not limited to galactose receptor, mannose receptor, scavengerreceptor, low-density lipoprotein receptor, HARE, CD44, IFNαreceptor,collagen type VI receptor, 6-phosphate/insulin-like growth factor 2receptor, platelet-derived growth factor receptor β, RBP receptor, αVβ3integrin receptor, ASGP receptor, glycyrrhetinic acid/glycyrrhizinreceptor, PPAR, Heparan sulfate glycosaminoglycan receptor, CXC receptortype 4, glycyrrhetinic acid receptor, HBVP receptor, HDL receptor,scavenger receptor class B member 1 LDL receptor or combination thereof.

Target Antigen Binding Domain

The trispecific antigen-binding proteins and bispecific antigen-bindingproteins described herein comprise a domain that binds to a targetantigen. A target antigen is involved in and/or associated with adisease, disorder or condition, e.g., cancer. In some embodiments, atarget antigen is a tumor antigen. In some embodiments, the targetantigen is NY-ESO-1, SSX-2, Sp 17, AFP, Glypican-3, Gpa33, Annexin-A2,WT1, PSMA, Midkine, PRAME, Survivin, MUC-1. P53, CEA, RAS, Hsp70, Hsp27,squamous cell carcinoma antigen (SCCA), GP73, TAG-72, or a protein inthe MAGE family.

In some embodiments, a target antigen is one found on a non-liver tumorcell that has metastasized into the liver. In some embodiments, abispecific antigen-binding protein or trispecific antigen bindingprotein comprises a target antigen binding domain specific for groupCD19, CD123, CD22, CD30, CD171, CS-1, C-type lectin-like molecule-1,CD33, epidermal growth factor receptor variant III (EGFRvIII),ganglioside G2 (GD2), ganglioside GD3, TNF receptor family member B cellmaturation (BCMA), Tn antigen ((Tn Ag) or (GaINAca-Ser/Thr)),prostate-specific membrane antigen (PSMA), Receptor tyrosine kinase-likeorphan receptor 1 (ROR1), Fms-Like Tyrosine Kinase 3 (FLT3),Tumor-associated glycoprotein 72 (TAG72), CD38, CD44v6, Carcinoembryonicantigen (CEA), Epithelial cell adhesion molecule (EPCAM), B7H3 (CD276),KIT (CD117), Interleukin-13 receptor subunit alpha-2, mesothelin,Interleukin 11 receptor alpha (IL-11Ra), prostate stem cell antigen(PSCA), Protease Serine 21, vascular endothelial growth factor receptor2 (VEGFR2), Lewis(Y) antigen, CD24, Platelet-derived growth factorreceptor beta (PDGFR-beta), Stage-specific embryonic antigen-4 (SSEA-4),CD20, Folate receptor alpha, HER2, HER3, Mucin 1, cell surfaceassociated (MUC1), epidermal growth factor receptor (EGFR), neural celladhesion molecule (NCAM), Prostase, prostatic acid phosphatase (PAP),elongation factor 2 mutated (ELF2M), Ephrin B2, fibroblast activationprotein alpha (FAP), insulin-like growth factor 1 receptor (IGF-Ireceptor), carbonic anhydrase IX (CAIX), Proteasome (Prosome, Macropain)Subunit, Beta Type, 9 (LMP2), glycoprotein 100 (gp100), oncogene fusionprotein consisting of breakpoint cluster region (BCR) and Abelson murineleukemia viral oncogene homolog 1 (Abl) (bcr-abl), tyrosinase, ephrintype-A receptor 2 (EphA2), Fucosyl GM1, sialyl Lewis adhesion molecule(sLe), ganglioside GM3, transglutaminase 5 (TGS5), high molecularweight-melanoma-associated antigen (HMWMAA), o-acetyl-GD2 ganglioside(OAcGD2), Folate receptor beta, tumor endothelial marker 1 (TEM1/CD248),tumor endothelial marker 7-related (TEM7R), claudin 6 (CLDN6), claudin18.2 (CLDN18.2), thyroid stimulating hormone receptor (TSHR), Gprotein-coupled receptor class C group 5, member D (GPRC5D), chromosomeX open reading frame 61 (CXORF61), CD97, or CD179a. In some embodiments,a target antigen is an antigen associated with a viral disease, e.g., aviral antigen. In some embodiments, a target antigen is a hepatitis A,hepatitis B, hepatitis C, hepatitis D or hepatitis E antigen.

The design of the trispecific antigen-binding proteins described hereinallows the binding domain to a liver target antigen to be flexible inthat the binding domain to a liver target antigen can be any type ofbinding domain, including but not limited to, domains from a monoclonalantibody, a polyclonal antibody, a recombinant antibody, a humanantibody, a humanized antibody. In some embodiments, the binding domainto a liver target antigen is a single chain variable fragments (scFv),single-domain antibody such as a heavy chain variable domain (VH), alight chain variable domain (VL) and a variable domain (VHH) of camelidderived single domain antibody. In other embodiments, the binding domainto a liver target antigen is a non-Ig binding domain, i.e., antibodymimetic, such as anticalins, affitins, affibody molecules, affimers,affitins, alphabodies, avimers, DARPins, fynomers, kunitz domainpeptides, and monobodies. In further embodiments, the binding domain toa liver target antigen is a ligand or peptide that binds to orassociates with a target antigen.

3.7 PAH

In some embodiments, the present invention provides methods andcompositions for delivering circRNA encoding PAH to a subject for thetreatment of phenylketonuria (PKU). A suitable PAH circRNA encodes anyfull length, fragment or portion of a PAH protein which can besubstituted for naturally-occurring PAH protein activity and/or reducethe intensity, severity, and/or frequency of one or more symptomsassociated with PKU.

In some embodiments, a suitable RNA sequence for the present inventioncomprises a circRNA sequence encoding human PAH protein.

In some embodiments, a suitable RNA sequence may be an RNA sequence thatencodes a homolog or an analog of human PAH. As used herein, a homologor an analog of human PAH protein may be a modified human PAH proteincontaining one or more amino acid substitutions, deletions, and/orinsertions as compared to a wild-type or naturally-occurring human PAHprotein while retaining substantial PAH protein activity.

The present invention may be used to treat a subject who is sufferingfrom or susceptible to Phenylketonuria (PKU). PKU is an autosomalrecessive metabolic genetic disorder characterized by a mutation in thegene for the hepatic enzyme phenylalanine hydroxylase (PAH), renderingit nonfunctional. PAH is necessary to metabolize the amino acidphenylalanine (Phe) to the amino acid tyrosine (Tyr). When PAH activityis reduced, phenylalanine accumulates and is converted intophenylpyruvate (also known as phenylketone) which can be detected in theurine.

Phenylalanine is a large, neutral amino acid (LNAA). LNAAs compete fortransport across the blood-brain barrier (BBB) via the large neutralamino acid transporter (LNAAT). Excess Phe in the blood saturates thetransporter and tends to decrease the levels of other LNAAs in thebrain. Because several of these other amino acids are necessary forprotein and neurotransmitter synthesis, Phe buildup hinders thedevelopment of the brain, and can cause mental retardation.

In addition to hindered brain development, the disease can presentclinically with a variety of symptoms including seizures, albinismhyperactivity, stunted growth, skin rashes (eczema), microcephaly,and/or a “musty” odor to the baby's sweat and urine, due tophenylacetate, one of the ketones produced). Untreated children aretypically normal at birth, but have delayed mental and social skills,have a head size significantly below normal, and often demonstrateprogressive impairment of cerebral function. As the child grows anddevelops, additional symptoms including hyperactivity, jerking movementsof the arms or legs, EEG abnormalities, skin rashes, tremors, seizures,and severe learning disabilities tend to develop. However, PKU iscommonly included in the routine newborn screening panel of mostcountries that is typically performed 2-7 days after birth.

If PKU is diagnosed early enough, an affected newborn can grow up withrelatively normal brain development, but only by managing andcontrolling Phe levels through diet, or a combination of diet andmedication. All PKU patients must adhere to a special diet low in Phefor optimal brain development. The diet requires severely restricting oreliminating foods high in Phe, such as meat, chicken, fish, eggs, nuts,cheese, legumes, milk and other dairy products. Starchy foods, such aspotatoes, bread, pasta, and corn, must be monitored. Infants may stillbe breastfed to provide all of the benefits of breastmilk, but thequantity must also be monitored and supplementation for missingnutrients will be required. The sweetener aspartame, present in manydiet foods and soft drinks, must also be avoided, as aspartame containsphenylalanine.

Throughout life, patients can use supplementary infant formulas, pillsor specially formulated foods to acquire amino acids and other necessarynutrients that would otherwise be deficient in a low-phenylalanine diet.Some Phe is required for the synthesis of many proteins and is requiredfor appropriate growth, but levels of it must be strictly controlled inPKU patients. Additionally, PKU patients must take supplements oftyrosine, which is normally derived from phenylalanine. Othersupplements can include fish oil, to replace the long chain fatty acidsmissing from a standard Phe-free diet and improve neurologicaldevelopment and iron or carnitine. Another potential therapy for PKU istetrahydrobiopterin (BH4), a cofactor for the oxidation of Phe that canreduce blood levels of Phe in certain patients. Patients who respond toBH4 therapy may also be able to increase the amount of natural proteinthat they can eat.

In some embodiments, the expression of PAH protein is detectable inliver, kidney, heart, spleen, serum, brain, skeletal muscle, lymphnodes, skin, and/or cerebrospinal fluid.

In some embodiments, administering the provided composition results inthe expression of a PAH protein level at or above about 100 ng/mg, about200 ng/mg, about 300 ng/mg, about 400 ng/mg, about 500 ng/mg, about 600ng/mg, about 700 ng/mg, about 800 ng/mg, about 900 ng/mg, about 1000ng/mg, about 1200 ng/mg or about 1400 ng/mg of total protein in theliver.

In some embodiments, the expression of the PAH protein is detectable 1to 96 hours after administration. For example, in some embodiments,expression of PAH protein is detectable 1 to 84 hours, 1 to 72 hours, 1to 60 hours, 1 to 48 hours, 1 to 36 hours, 1 to 24 hours, 1 to 12 hours,1 to 10 hours, 1 to 8 hours, 1 to 6 hours, 1 to 4 hours, 1 to 2 hours, 2to 96 hours, 2 to 84 hours, 2 to 72 hours, 2 to 60 hours, 2 to 48 hours,2 to 36 hours, 2 to 24 hours, 2 to 12 hours, 2 to 10 hours, 2 to 8hours, 2 to 6 hours, 2 to 4 hours, 4 to 96 hours, 4 to 84 hours, 4 to 72hours, 4 to 60 hours, 4 to 48 hours, 4 to 36 hours, 4 to 24 hours, 4 to12 hours, 4 to 10 hours, 4 to 8 hours, 4 to 6 hours, 6 to 96 hours, 6 to84 hours, 6 to 72 hours, 6 to 60 hours, 6 to 48 hours, 6 to 36 hours, 6to 24 hours, 6 to 12 hours, 6 to 10 hours, 6 to 8 hours, 8 to 96 hours,8 to 84 hours, 8 to 72 hours, 8 to 60 hours, 8 to 48 hours, 8 to 36hours, 8 to 24 hours, 8 to 12 hours, 8 to 10 hours, 10 to 96 hours, 10to 84 hours, 10 to 72 hours, 10 to 60 hours, 10 to 48 hours, 10 to 36hours, 10 to 24 hours, 10 to 12 hours, 12 to 96 hours, 12 to 84 hours,12 to 72 hours, 12 to 60 hours, 12 to 48 hours, 12 to 36 hours, 12 to 24hours, 24 to 96 hours, 24 to 84 hours, 24 to 72 hours, 24 to 60 hours,24 to 48 hours, 24 to 36 hours, 36 to 96 hours, 36 to 84 hours, 36 to 72hours, 36 to 60 hours, 36 to 48 hours, 48 to 96 hours, 48 to 84 hours,48 to 72 hours, 48 to 60 hours, 48 to 84 hours, 48 to 72 hours, 48 to 60hours, 60 to 96 hours, 60 to 84 hours, 60 to 72 hours, 72 hours to 96hours, 72 hours to 84 hours, or 84 hours to 96 hours afteradministration. For example, in certain embodiments, the expression ofthe PAH protein is detectable 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66,and/or 72 hours after the administration. In some embodiments, theexpression of the PAH protein is detectable 1 day to 7 days after theadministration. For example, in some embodiments, PAH protein isdetectable 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, and/or 7 daysafter the administration. In some embodiments, the expression of the PAHprotein is detectable 1 week to 8 weeks after the administration. Forexample, in some embodiments, the expression of the PAH protein isdetectable 1 week, 2 weeks, 3 weeks, and/or 4 weeks after theadministration. In some embodiments, the expression of the PAH proteinis detectable after a month after the administration.

3.8 CPS1

In some embodiments, the present invention provides methods andcompositions for delivering circRNA encoding CPS1 to a subject for thetreatment of CPS1 deficiency. A suitable CPS1 circRNA encodes any fulllength, fragment or portion of a CPS1 protein which can be substitutedfor naturally-occurring CPS1 protein activity and/or reduce theintensity, severity, and/or frequency of one or more symptoms associatedwith CPS1 deficiency.

In some embodiments, a suitable RNA sequence for the present inventioncomprises a circRNA sequence encoding human CPS1 protein.

In some embodiments, a suitable RNA sequence may be an RNA sequence thatencodes a homolog or an analog of human CPS1. As used herein, a homologor an analog of human CPS1 protein may be a modified human CPS1 proteincontaining one or more amino acid substitutions, deletions, and/orinsertions as compared to a wild-type or naturally-occurring human CPS1protein while retaining substantial CPS1 protein activity.

Carbamoyl phosphate synthetase I (CPS1) catalyzes the conversion ofammonia, bicarbonate and 2 ATP with formation of carbamoyl phosphate inthe first step of the urea cycle. It also plays a role in thebiosynthesis of arginine, which in turn is a substrate for thebiosynthesis of NO, e.g. in the case of an endotoxin shock (c.f. ShokoTabuchi et al., Regulation of Genes for Inducible Nitric Oxide Synthaseand Urea Cycle Enzymes in Rat Liver in Endotoxin Shock, Biochemical andBiophysical Research Communications 268, 221-224 (2000)). CPS 1 shouldbe distinguished from the cytosolic enzyme CPS 2, which likewise plays arole in the urea cycle but processes the substrate glutamine. It isknown that CPS 1 is localized in mitochondria and occurs in this form inlarge amounts in liver tissue (it accounts for 2-6% of total liverprotein). Its amino acid sequence and genetic localization have longbeen known (c.f. Haraguchi Y. et al., Cloning and sequence of a cDNAencoding human carbamyl phosphate synthetase I: molecular analysis ofhyperammonemia, Gene 1991 Nov. 1; 107 (2); 335-340; cf. also thepublication WO 03/089933 A1 of the Applicant). Regarding itsphysiological role, reference may be made to review articles such as,for example, H. M. Holder et al., Carbamoyl phosphate synthetase: anamazing biochemical odyssey from substrate to product, CMLS, Cell. Mol.Life Sci. 56 (1999) 507-522, and the literature referred to therein, andthe introduction to the publication by Mikiko Ozaki et al.,Enzyme-Linked Immunosorbent Assay of Carbamoylphosphate Synthetase I:Plasma Enzyme in Rat Experimental Hepatitis and Its Clearance, EnzymeProtein 1994, 95:48:213-221.

Carbamoyl phosphate synthetase I (CPS1) deficiency is a genetic disordercharacterized by a mutation in the gene for the enzyme Carbamoylphosphate synthetase I, affecting its ability to catalyze synthesis ofcarbamoyl phosphate from ammonia and bicarbonate. This reaction is thefirst step of the urea cycle, which is important in the removal ofexcess urea from cells. Defects in the CPS1 protein disrupt the ureacycle and prevent the liver from properly processing excess nitrogeninto urea.

In some embodiments, administering the provided composition results inthe expression of a CPS1 protein level at or above about 100 ng/mg,about 200 ng/mg, about 300 ng/mg, about 400 ng/mg, about 500 ng/mg,about 600 ng/mg, about 700 ng/mg, about 800 ng/mg, about 900 ng/mg,about 1000 ng/mg, about 1200 ng/mg or about 1400 ng/mg of total proteinin the liver.

In some embodiments, the expression of the CPS1 protein is detectable 1to 96 hours after administration. For example, in some embodiments,expression of CPS1 protein is detectable 1 to 84 hours, 1 to 72 hours, 1to 60 hours, 1 to 48 hours, 1 to 36 hours, 1 to 24 hours, 1 to 12 hours,1 to 10 hours, 1 to 8 hours, 1 to 6 hours, 1 to 4 hours, 1 to 2 hours, 2to 96 hours, 2 to 84 hours, 2 to 72 hours, 2 to 60 hours, 2 to 48 hours,2 to 36 hours, 2 to 24 hours, 2 to 12 hours, 2 to 10 hours, 2 to 8hours, 2 to 6 hours, 2 to 4 hours, 4 to 96 hours, 4 to 84 hours, 4 to 72hours, 4 to 60 hours, 4 to 48 hours, 4 to 36 hours, 4 to 24 hours, 4 to12 hours, 4 to 10 hours, 4 to 8 hours, 4 to 6 hours, 6 to 96 hours, 6 to84 hours, 6 to 72 hours, 6 to 60 hours, 6 to 48 hours, 6 to 36 hours, 6to 24 hours, 6 to 12 hours, 6 to 10 hours, 6 to 8 hours, 8 to 96 hours,8 to 84 hours, 8 to 72 hours, 8 to 60 hours, 8 to 48 hours, 8 to 36hours, 8 to 24 hours, 8 to 12 hours, 8 to 10 hours, 10 to 96 hours, 10to 84 hours, 10 to 72 hours, 10 to 60 hours, 10 to 48 hours, 10 to 36hours, 10 to 24 hours, 10 to 12 hours, 12 to 96 hours, 12 to 84 hours,12 to 72 hours, 12 to 60 hours, 12 to 48 hours, 12 to 36 hours, 12 to 24hours, 24 to 96 hours, 24 to 84 hours, 24 to 72 hours, 24 to 60 hours,24 to 48 hours, 24 to 36 hours, 36 to 96 hours, 36 to 84 hours, 36 to 72hours, 36 to 60 hours, 36 to 48 hours, 48 to 96 hours, 48 to 84 hours,48 to 72 hours, 48 to 60 hours, 48 to 84 hours, 48 to 72 hours, 48 to 60hours, 60 to 96 hours, 60 to 84 hours, 60 to 72 hours, 72 hours to 96hours, 72 hours to 84 hours, or 84 hours to 96 hours afteradministration. For example, in certain embodiments, the expression ofthe CPS1 protein is detectable 6, 12, 18, 24, 30, 36, 42, 48, 54, 60,66, and/or 72 hours after the administration. In some embodiments, theexpression of the CPS1 protein is detectable 1 day to 7 days after theadministration. For example, in some embodiments, CPS1 protein isdetectable 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, and/or 7 daysafter the administration. In some embodiments, the expression of theCPS1 protein is detectable 1 week to 8 weeks after the administration.For example, in some embodiments, CPS1 protein is detectable 1 week, 2weeks, 3 weeks, and/or 4 weeks after the administration. In someembodiments, the expression of the CPS1 protein is detectable after amonth after the administration.

In some embodiments, administering of the composition results in reducedammonia levels in a subject as compared to baseline levels beforetreatment. Typically, baseline levels are measured in the subjectimmediately before treatment. Typically, ammonia levels are measured ina biological sample. Suitable biological samples include, for example,whole blood, plasma, serum, urine or cerebral spinal fluid.

In some embodiments, administering the composition results in reducedammonia levels in a biological sample (e.g., a serum, plasma, or urinesample) by at least about 10%, at least about 15%, at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 55%,at least about 60%, at least about 65%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,or at least about 95% as compared to baseline levels in a subjectimmediately before treatment.

In some embodiments, administering the composition provided hereinresults in reduced ammonia levels in plasma or serum as compared tobaseline ammonia levels in a subject immediately before treatment. Insome embodiments, administering the provided composition results inreduced ammonia levels in plasma or serum as compared to the ammonialevels in subjects who are not treated. In some embodiments,administering the composition results in reduction of ammonia levels toabout 3000 μmol/L or less, about 2750 μmol/L or less, about 2500 μmol/Lor less, about 2250 μmol/L or less, about 2000 μmol/L or less, about1750 μmol/L or less, about 1500 μmol/L or less, about 1250 μmol/L orless, about 1000 μmol/L or less, about 750 μmol/L or less, about 500μmol/L or less, about 250 μmol/L or less, about 100 μmol/L or less orabout 50 μmol/L or less in the plasma or serum of the subject. In aparticular embodiment, administering the composition results inreduction of ammonia levels to about 50 μmol/L or less in the plasma orserum.

3.9 ADAMTS13

In some embodiments, the present invention provides methods andcompositions for delivering circRNA encoding ADAMTS13 to a subject forthe treatment of thrombotic thrombocytopenic purpura (TTP). A suitableADAMTS13 circRNA encodes any full length ADAMTS 13 protein, orfunctional fragment or portion thereof, which can be substituted fornaturally-occurring ADAMTS 13 protein and/or reduce the intensity,severity, and/or frequency of one or more symptoms associated with TTP.

In some embodiments, the RNA sequence of the present invention comprisesa circRNA sequence encoding human ADAMTS13 protein.

In some embodiments, the RNA sequence may be an RNA sequence thatencodes a homolog or an analog of human ADAMTS13. As used herein, ahomolog or an analog of human ADAMTS 13 protein may be a modified humanADAMTS13 protein containing one or more amino acid substitutions,deletions, and/or insertions as compared to a wild-type ornaturally-occurring human ADAMTS13 protein while retaining substantialADAMTS13 protein activity.

The ADAMTS13 enzyme cleaves von Willebrand factor, which, in itsun-cleaved form, interacts with platelets and causes them to sticktogether and adhere to the walls of blood vessels, forming clots.Defects in ADAMTS13 are associated with TTP.

In some embodiments, administering the provided composition results inthe expression of a ADAMTS13 protein level at or above about 100 ng/mg,about 200 ng/mg, about 300 ng/mg, about 400 ng/mg, about 500 ng/mg,about 600 ng/mg, about 700 ng/mg, about 800 ng/mg, about 900 ng/mg,about 1000 ng/mg, about 1200 ng/mg or about 1400 ng/mg of total proteinin the liver.

In some embodiments, the expression of the ADAMTS13 protein isdetectable 1 to 96 hours after administration. For example, in someembodiments, expression of ADAMTS13 protein is detectable 1 to 84 hours,1 to 72 hours, 1 to 60 hours, 1 to 48 hours, 1 to 36 hours, 1 to 24hours, 1 to 12 hours, 1 to 10 hours, 1 to 8 hours, 1 to 6 hours, 1 to 4hours, 1 to 2 hours, 2 to 96 hours, 2 to 84 hours, 2 to 72 hours, 2 to60 hours, 2 to 48 hours, 2 to 36 hours, 2 to 24 hours, 2 to 12 hours, 2to 10 hours, 2 to 8 hours, 2 to 6 hours, 2 to 4 hours, 4 to 96 hours, 4to 84 hours, 4 to 72 hours, 4 to 60 hours, 4 to 48 hours, 4 to 36 hours,4 to 24 hours, 4 to 12 hours, 4 to 10 hours, 4 to 8 hours, 4 to 6 hours,6 to 96 hours, 6 to 84 hours, 6 to 72 hours, 6 to 60 hours, 6 to 48hours, 6 to 36 hours, 6 to 24 hours, 6 to 12 hours, 6 to 10 hours, 6 to8 hours, 8 to 96 hours, 8 to 84 hours, 8 to 72 hours, 8 to 60 hours, 8to 48 hours, 8 to 36 hours, 8 to 24 hours, 8 to 12 hours, 8 to 10 hours,10 to 96 hours, 10 to 84 hours, 10 to 72 hours, 10 to 60 hours, 10 to 48hours, 10 to 36 hours, 10 to 24 hours, 10 to 12 hours, 12 to 96 hours,12 to 84 hours, 12 to 72 hours, 12 to 60 hours, 12 to 48 hours, 12 to 36hours, 12 to 24 hours, 24 to 96 hours, 24 to 84 hours, 24 to 72 hours,24 to 60 hours, 24 to 48 hours, 24 to 36 hours, 36 to 96 hours, 36 to 84hours, 36 to 72 hours, 36 to 60 hours, 36 to 48 hours, 48 to 96 hours,48 to 84 hours, 48 to 72 hours, 48 to 60 hours, 48 to 84 hours, 48 to 72hours, 48 to 60 hours, 60 to 96 hours, 60 to 84 hours, 60 to 72 hours,72 hours to 96 hours, 72 hours to 84 hours, or 84 hours to 96 hoursafter administration. For example, in certain embodiments, theexpression of the ADAMTS13 protein is detectable 6, 12, 18, 24, 30, 36,42, 48, 54, 60, 66, and/or 72 hours after the administration. In someembodiments, the expression of the ADAMTS13 protein is detectable 1 dayto 7 days after the administration. For example, in some embodiments,ADAMTS13 protein is detectable 1 day, 2 days, 3 days, 4 days, 5 days, 6days, and/or 7 days after the administration. In some embodiments, theexpression of the ADAMTS13 protein is detectable 1 week to 8 weeks afterthe administration. For example, in some embodiments, ADAMTS13 proteinis detectable 1 week, 2 weeks, 3 weeks, and/or 4 weeks after theadministration. In some embodiments, the expression of the ADAMTS13protein is detectable after a month after the administration.

In some embodiments, administering the composition results in reducedvon Willebrand factor (vWF) levels in a subject as compared to baselinevWR levels before treatment. Typically, the baseline levels are measuredin the subject immediately before treatment. Typically, vWF levels aremeasured in a biological sample. Suitable biological samples include,for example, whole blood, plasma or serum.

In some embodiments, administering the composition results in reducedvWF levels in a biological sample taken from the subject by at leastabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% as compared tobaseline vWF levels immediately before treatment. In some embodiments,administering the composition results in reduced plasma vWF levels inthe subject to less than about 2000 μM, 1500 μM, 1000 μM, 750 μM, 500μM, 250 μM, 100 μM, 90 μM, 80 μM, 70 μM, 60 μM, 50 μM, 40 μM, or 30 μm.

In some embodiments, administering the provided composition results inreduced vWF levels in plasma or serum samples taken from the subject ascompared to baseline vWF levels immediately before treatment. In someembodiments, administering the provided composition results in reducedvWF levels in plasma or serum as compared to vWF levels in subjects whoare not treated. In some embodiments, administering the compositionresults in reduction of vWF levels to about 3000 μmol/L or less, about2750 μmol/L or less, about 2500 μmol/L or less, about 2250 μmol/L orless, about 2000 μmol/L or less, about 1750 μmol/L or less, about 1500μmol/L or less, about 1250 μmol/L or less, about 1000 μmol/L or less,about 750 μmol/L or less, about 500 μmol/L or less, about 250 μmol/L orless, about 100 μmol/L or less or about 50 μmol/L or less in the plasmaor serum. In a particular embodiment, administering the compositionresults in reduction of vWF levels to about 50 μmol/L or less in theplasma or serum

4. Production of Polynucleotides

The vectors provided herein can be made using standard techniques ofmolecular biology. For example, the various elements of the vectorsprovided herein can be obtained using recombinant methods, such as byscreening cDNA and genomic libraries from cells, or by deriving thepolynucleotides from a vector known to include the same.

The various elements of the vectors provided herein can also be producedsynthetically, rather than cloned, based on the known sequences. Thecomplete sequence can be assembled from overlapping oligonucleotidesprepared by standard methods and assembled into the complete sequence.See, e.g., Edge, Nature (1981) 292:756; Nambair et al., Science (1984)223: 1299; and Jay et al., J. Biol. Chem. (1984) 259:631 1.

Thus, particular nucleotide sequences can be obtained from vectorsharboring the desired sequences or synthesized completely or in partusing various oligonucleotide synthesis techniques known in the art,such as site-directed mutagenesis and polymerase chain reaction (PCR)techniques where appropriate. One method of obtaining nucleotidesequences encoding the desired vector elements is by annealingcomplementary sets of overlapping synthetic oligonucleotides produced ina conventional, automated polynucleotide synthesizer, followed byligation with an appropriate DNA ligase and amplification of the ligatednucleotide sequence via PCR. See, e.g., Jayaraman et al., Proc. Natl.Acad. Sci. USA (1991) 88:4084-4088. Additionally,oligonucleotide-directed synthesis (Jones et al., Nature (1986)54:75-82), oligonucleotide directed mutagenesis of preexistingnucleotide regions (Riechmann et al., Nature (1988) 332:323-327 andVerhoeyen et al., Science (1988) 239: 1534-1536), and enzymaticfilling-in of gapped oligonucleotides using T4 DNA polymerase (Queen etal., Proc. Natl. Acad. Sci. USA (1989) 86: 10029-10033) can be used.

The precursor RNA provided herein can be generated by incubating avector provided herein under conditions permissive of transcription ofthe precursor RNA encoded by the vector. For example, in someembodiments a precursor RNA is synthesized by incubating a vectorprovided herein that comprises an RNA polymerase promoter upstream ofits 5′ duplex forming region and/or expression sequence with acompatible RNA polymerase enzyme under conditions permissive of in vitrotranscription. In some embodiments, the vector is incubated inside of acell by a bacteriophage RNA polymerase or in the nucleus of a cell byhost RNA polymerase II.

In certain embodiments, provided herein is a method of generatingprecursor RNA by performing in vitro transcription using a vectorprovided herein as a template (e.g., a vector provided herein with a RNApolymerase promoter positioned upstream of the 5′ homology region).

In certain embodiments, the resulting precursor RNA can be used togenerate circular RNA (e.g., a circular RNA polynucleotide providedherein) by incubating it in the presence of magnesium ions and guanosinenucleotide or nucleoside at a temperature at which RNA circularizationoccurs (e.g., between 20° C. and 60° C.).

Thus, in certain embodiments provided herein is a method of makingcircular RNA. In certain embodiments, the method comprises synthesizingprecursor RNA by transcription (e.g., run-off transcription) using avector provided herein (e.g., a vector comprising, in the followingorder, a 5′ homology region, a 3′ group I intron fragment, a firstspacer, an Internal Ribosome Entry Site (IRES), an expression sequence,a second spacer, a 5′ group I intron fragment, and a 3′ homology region)as a template, and incubating the resulting precursor RNA in thepresence of divalent cations (e.g., magnesium ions) and GTP such that itcircularizes to form circular RNA. In some embodiments, the precursorRNA disclosed herein is capable of circularizing in the absence ofmagnesium ions and GTP and/or without the step of incubation withmagnesium ions and GTP. It has been discovered that circular RNA hasreduced immunogenicity relative to a corresponding mRNA, at leastpartially because the mRNA contains an immunogenic 5′ cap. Whentranscribing a DNA vector from certain promoters (e.g., a T7 promoter)to produce a precursor RNA, it is understood that the 5′ end of theprecursor RNA is G. To reduce the immunogenicity of a circular RNAcomposition that contains a low level of contaminant linear mRNA, anexcess of GMP relative to GTP can be provided during transcription suchthat most transcripts contain a 5′ GMP, which cannot be capped.Therefore, in some embodiments, transcription is carried out in thepresence of an excess of GMP. In some embodiments, transcription iscarried out where the ratio of GMP concentration to GTP concentration iswithin the range of about 3:1 to about 15:1, for example, about 3:1 toabout 10:1, about 3:1 to about 5:1, about 3:1, about 4:1, or about 5:1.

In some embodiments, a composition comprising circular RNA has beenpurified. Circular RNA may be purified by any known method commonly usedin the art, such as column chromatography, gel filtrationchromatography, and size exclusion chromatography. In some embodiments,purification comprises one or more of the following steps: phosphatasetreatment, HPLC size exclusion purification, and RNase R digestion. Insome embodiments, purification comprises the following steps in order:RNase R digestion, phosphatase treatment, and HPLC size exclusionpurification. In some embodiments, purification comprises reverse phaseHPLC. In some embodiments, a purified composition contains less doublestranded RNA, DNA splints, triphosphorylated RNA, phosphatase proteins,protein ligases, capping enzymes and/or nicked RNA than unpurified RNA.In some embodiments, a purified composition is less immunogenic than anunpurified composition. In some embodiments, immune cells exposed to apurified composition produce less IFN-β1, RIG-I, IL-2, IL-6, IFNγ,and/or TNFα than immune cells exposed to an unpurified composition.

5. Ionizable Lipids

In certain embodiments disclosed herein are ionizable lipids that may beused as a component of a transfer vehicle to facilitate or enhance thedelivery and release of circular RNA to one or more target cells (e.g.,by permeating or fusing with the lipid membranes of such target cells).In certain embodiments, an ionizable lipid comprises one or morecleavable functional groups (e.g., a disulfide) that allow, for example,a hydrophilic functional head-group to dissociate from a lipophilicfunctional tail-group of the compound (e.g., upon exposure to oxidative,reducing or acidic conditions), thereby facilitating a phase transitionin the lipid bilayer of the one or more target cells.

In some embodiments, an ionizable lipid is a lipid as described ininternational patent application PCT/US2018/058555.

In some of embodiments, a cationic lipid has the following formula:

wherein:

-   -   R₁ and R₂ are either the same or different and independently        optionally substituted C₁₀-C₂₄ alkyl, optionally substituted        C₁₀-C₂₄ alkenyl, optionally substituted C₁₀-C₂₄ alkynyl, or        optionally substituted C₁₀-C₂₄ acyl;    -   R₃ and R₄ are either the same or different and independently        optionally substituted C₁-C₆alkyl, optionally substituted C₂-C₆        alkenyl, or optionally substituted C₂-C₆ alkynyl or R₃ and R₄        may join to form an optionally substituted heterocyclic ring of        4 to 6 carbon atoms and 1 or 2 heteroatoms chosen from nitrogen        and oxygen;    -   R₅ is either absent or present and when present is hydrogen or        C₁-C₆ alkyl; m, n, and p are either the same or different and        independently either 0 or 1 with the proviso that m, n, and p        are not simultaneously 0; q is 0, 1, 2, 3, or 4; and    -   Y and Z are either the same or different and independently O, S.        or NH.

In one embodiment, R₁ and R₂ are each linoleyl, and the amino lipid is adilinoleyl amino lipid.

In one embodiment, the amino lipid is a dilinoleyl amino lipid.

In various other embodiments, a cationic lipid has the followingstructure:

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomerthereof, wherein:

-   -   R₁ and R₂ are each independently selected from the group        consisting of H and C₁-C₃ alkyls; and    -   R₃ and R₄ are each independently an alkyl group having from        about 10 to about 20 carbon atoms, wherein at least one of R₃        and R₄ comprises at least two sites of unsaturation.

In some embodiments, R₃ and R₄ are each independently selected fromdodecadienyl, tetradecadienyl, hexadecadienyl, linoleyl, andicosadienyl. In an embodiment, R₃ and R₄ and are both linoleyl. In someembodiments, R₃ and/or R₄ may comprise at least three sites ofunsaturation (e.g., R₃ and/or R₄ may be, for example, dodecatrienyl,tetradectrienyl, hexadecatrienyl, linolenyl, and icosatrienyl).

In some embodiments, a cationic lipid has the following structure:

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomerthereof, wherein:

-   -   R₁ and R₂ are each independently selected from H and C₁-C₃        alkyls;    -   R₃ and R₄ are each independently an alkyl group having from        about 10 to about 20 carbon atoms, wherein at least one of R₃        and R₄ comprises at least two sites of unsaturation.

In one embodiment, R₃ and R₄ are the same, for example, in someembodiments R₃ and R₄ are both linoleyl (C₁₈-alkyl). In anotherembodiment, R₃ and R₄ are different, for example, in some embodiments,R₃ is tetradectrienyl (C₁₄-alkyl) and R₄ is linoleyl (C₁₈-alkyl). In apreferred embodiment, the cationic lipid(s) of the present invention aresymmetrical, i.e., R₃ and R₄ are the same. In another preferredembodiment, both R₃ and R₄ comprise at least two sites of unsaturation.In some embodiments, R₃ and R₄ are each independently selected fromdodecadienyl, tetradecadienyl, hexadecadienyl, linoleyl, andicosadienyl. In an embodiment, R₃ and R₄ are both linoleyl. In someembodiments, R₃ and/or R₄ comprise at least three sites of unsaturationand are each independently selected from dodecatrienyl, tetradectrienyl,hexadecatrienyl, linolenyl, and icosatrienyl.

In various embodiments, a cationic lipid has the formula:

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomerthereof, wherein:

-   -   X_(aa) is a D- or L-amino acid residue having the formula        —NR^(N)—CR¹R²—C(C═O)—, or a peptide or a peptide of amino acid        residues having the formula —{NR^(N)—CR¹R²—C(C═O)}_(n)—, wherein        n is an integer from 2 to 20;    -   R¹ is independently, for each occurrence, a non-hydrogen or a        substituted or unsubstituted side chain of an amino acid;    -   R² and R^(N) are independently, for each occurrence, hydrogen,        an organic group consisting of carbon, oxygen, nitrogen, sulfur,        and hydrogen atoms, or any combination of the foregoing, and        having from 1 to 20 carbon atoms, C₍₁₋₅₎alkyl, cycloalkyl,        cycloalkylalkyl, C₍₁₋₅₎alkenyl, C₍₁₋₅₎alkynyl, C₍₁₋₅₎alkanoyl,        C₍₁₋₅₎alkanoyloxy, C₍₁₋₅₎alkoxy, C₍₁₋₅₎alkoxy-C₍₁₋₅₎alkyl,        C₍₁₋₅₎alkoxy-C₍₁₋₅₎alkoxy, C₍₁₋₅₎alkyl-amino-C₍₁₋₅₎alkyl-,        C₍₁₋₅₎dialkyl-amino-C₍₁₋₅₎alkyl-, nitro-C₍₁₋₅₎alkyl,        cyano-C₍₁₋₅₎alkyl, aryl-C₍₁₋₅₎alkyl, 4-biphenyl-C₍₁₋₅₎alkyl,        carboxyl, or hydroxyl;    -   Z is —NH—, —O—, —S—, —CH₂S—, —CH₂S(O)—, or an organic linker        consisting of 1-40 atoms selected from hydrogen, carbon, oxygen,        nitrogen, and sulfur atoms (preferably, Z is —NH— or —O—);    -   R^(x) and R^(y) are, independently, (i) a lipophilic tail        derived from a lipid (which can be naturally occurring or        synthetic), e.g., a phospholipid, a glycolipid, a        triacylglycerol, a glycerophospholipid, a sphingolipid, a        ceramide, a sphingomyelin, a cerebroside, or a ganglioside,        wherein the tail optionally includes a steroid; (ii) an amino        acid terminal group selected from hydrogen, hydroxyl, amino, and        an organic protecting group; or (iii) a substituted or        unsubstituted C₍₃₋₂₂₎alkyl, C₍₆₋₁₂₎cycloalkyl,        C₍₆₋₁₂₎cycloalkyl-C₍₃₋₂₂₎alkyl, C₍₃₋₂₂₎alkenyl, C₍₃₋₂₂₎alkynyl,        C₍₃₋₂₂₎alkoxy, or C₍₆₋₁₂₎-alkoxy C₍₃₋₂₂₎alkyl;

In some embodiments, one of R^(x) and R^(y) is a lipophilic tail asdefined above and the other is an amino acid terminal group. In someembodiments, both R^(x) and R^(y) are lipophilic tails.

In some embodiments, at least one of R^(x) and R^(y) is interrupted byone or more biodegradable groups (e.g., —OC(O)—, —C(O)O—, —SC(O)—,—C(O)S—, —OC(S)—, —C(S)O—, —S—S—, —C(O)(NR⁵)—, —N(R⁵)C(O)—, —C(S)(NR⁵)—,—N(R⁵)C(O)—, —N(R⁵)C(O)N(R⁵)—, —OC(O)O—, —OSi(R⁵)₂O—,—C(O)(CR³R⁴)C(O)O—, —OC(O)(CR³R⁴)C(O)—, or

In some embodiments, R¹¹ is a C₂-C₈alkyl or alkenyl.

In some embodiments, each occurrence of R⁵ is, independently, H oralkyl.

In some embodiments, each occurrence of R³ and R⁴ are, independently H,halogen, OH, alkyl, alkoxy, —NH₂, alkylamino, or dialkylamino; or R₃ andR₄, together with the carbon atom to which they are directly attached,form a cycloalkyl group. In some particular embodiments, each occurrenceof R³ and R⁴ are, independently H or C₁-C₄alkyl.

In some embodiments, R^(x) and R^(y) each, independently, have one ormore carbon-carbon double bonds.

In some embodiments, the cationic lipid is one of the following:

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomerthereof, wherein:

-   -   R¹ and R₂ are each independently alkyl, alkenyl, or alkynyl,        each of which can optionally substituted;    -   R₃ and R₄ are each independently a C₁-C₆ alkyl, or R₃ and R₄ are        taken together to form an optionally substituted heterocyclic        ring.

A representative useful dilinoleyl amino lipid has the formula:

wherein n is 0, 1, 2, 3, or 4.

In one embodiment, a cationic lipid is DLin-K-DMA. In one embodiment, acationic lipid is DLin-KC2-DMA (DLin-K-DMA above, wherein n is 2).

In one embodiment, a cationic lipid has the following structure:

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomerthereof, wherein:

-   -   R₁ and R₂ are each independently for each occurrence optionally        substituted C₁₀-C₃₀ alkyl, optionally substituted C₁₀-C₃₀        alkenyl, optionally substituted C₁₀-C₃₀ alkynyl or optionally        substituted C₁₀-C₃₀ acyl;    -   R₃ is H, optionally substituted C₂-C₁₀ alkyl, optionally        substituted C₂-C₁₀ alkenyl, optionally substituted C₂-C₁₀        alkylyl, alkylhetrocycle, alkylpbosphate, alkylphosphorothioate,        alkylphosphorodithioate, alkylphosphonate, alkylamine,        hydroxyalkyl, ω-aminoalkyl, ω-(substituted)aminoalkyl,        ω-phosphoalkyl, ω-thiophosphoalkyl, optionally substituted        polyethylene glycol (PEG, mw 100-40K), optionally substituted        mPEG (mw 120-40K), heteroaryl, or heterocycle, or a linker        ligand, for example, in some embodiments, R₃ is        (CH₃)₂N(CH₂)_(n)—, wherein n is 1, 2, 3 or 4;    -   E is O, S, N(Q), C(O), OC(O), C(O)O, N(Q)C(O), C(O)N(Q),        (Q)N(CO)O, O(CO)N(Q), S(O), NS(O)₂N(O), S(O)₂, N(Q)S(O)₂, SS,        O═N, aryl, heteroaryl, cyclic or heterocycle, for example        —C(O)O, wherein - is a point of connection to R₃; and    -   Q is H, alkyl, ω-aminoalkyl, ω-(substituted)aminoalkyl,        ω-phosphoalkyl or ω-thiophosphoalkyl.

In one specific embodiment, the cationic lipid of Embodiments 1, 2, 3, 4or 5 has the following structure:

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomerthereof, wherein:

-   -   E is O, S, N(Q), C(O), N(Q)C(O), C(O)N(Q), (Q)N(CO)O, O(CO)N(Q).        S(O), NS(O)₂N(Q), S(O)₂, N(Q)S(O)₂, SS, O═N, aryl, heteroaryl,        cyclic or heterocycle;    -   Q is H, alkyl, ω-aminoalkyl, ω-(substituted)aminoalkyl,        ω-phosphoalkyl or ω-thiophosphoalkyl;    -   R₁ and R₂ and R_(x) are each independently for each occurrence        H, optionally substituted C₁-C₁₀ alkyl, optionally substituted        C₁₀-C₃₀ alkyl, optionally substituted C₁₀-C₃₀ alkenyl,        optionally substituted C₁₀-C₃₀ alkynyl, optionally substituted        C₁₀-C₃₀ acyl, or linker-ligand, provided that at least one of        R₁, R₂ and R_(x) is not H;    -   R₃ is optionally substituted C₁-C₁₀ alkyl, optionally        substituted C₂-C₁₀ alkenyl, optionally substituted C₂-C₁₀        alkynyl, alkylhetrocycle, alkylphosphate, alkylphosphorothioate,        alkylphosphorodithioate, alkylphosphonate, alkylamine,        hydroxyalkyl, ω-aminoalkyl, ω-(substituted)aminoalkyl,        ω-phosphoalkyl, ω-thiophosphoalkyl, optionally substituted        polyethylene glycol (PEG, mw 100-40K), optionally substituted        mPEG (mw 120-40K), heteroaryl, or heterocycle, or linker-ligand;        and    -   n is 0, 1, 2, or 3

In one embodiment, the cationic lipid of Embodiments 1, 2, 3, 4 or 5 hasthe structure of Formula I:

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomerthereof, wherein:

-   -   one of L¹ or L² is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)_(x)—,        —S—S—, —C(═O)S—, SC(═O)—, —NR^(a)C(═O)—, —C(═O)NR^(a)—,        NR^(a)C(═O)NR^(a)—, —OC(═O)NR^(a)— or —NR^(a)C(═O)O—, and the        other of L¹ or L² is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—,        —S(O)_(x)—, —S—S—, —C(═O)S—, SC(═O)—, —NR^(a)C(═O)—,        —C(═O)NR^(a)—, NR^(a)C(═O)NR^(a)—, —OC(═O)NR^(a)— or        —NR^(a)C(═O)O— or a direct bond;    -   R^(a) is H or C₁-C₁₂ alkyl;    -   R^(1a) and R^(1b) are, at each occurrence, independently        either (a) H or C₁-C₁₂ alkyl, or (b) R^(1a) is H or C₁-C₁₂        alkyl, and R^(1b) together with the carbon atom to which it is        bound is taken together with an adjacent R^(1b) and the carbon        atom to which it is bound to form a carbon-carbon double bond;    -   R^(2a) and R^(2b) are, at each occurrence, independently        either (a) H or C₁-C₁₂ alkyl, or (b) R^(2a) is H or C₁-C₁₂        alkyl, and R^(2b) together with the carbon atom to which it is        bound is taken together with an adjacent R^(2b) and the carbon        atom to which it is bound to form a carbon-carbon double bond;    -   R^(3a) and R^(3b) are, at each occurrence, independently        either (a) H or C₁-C₁₂ alkyl, or (b) R^(3a) is H or C₁-C₁₂        alkyl, and R^(3b) together with the carbon atom to which it is        bound is taken together with an adjacent R^(3b) and the carbon        atom to which it is bound to form a carbon-carbon double bond;    -   R^(4a) and R^(4b) are, at each occurrence, independently        either (a) H or C₁-C₁₂ alkyl, or (b) R^(4a) is H or C₁-C₁₂        alkyl, and R^(4b) together with the carbon atom to which it is        bound is taken together with an adjacent R^(4b) and the carbon        atom to which it is bound to form a carbon-carbon double bond;    -   R⁵ and R⁶ are each independently methyl or cycloalkyl;    -   R⁷ is, at each occurrence, independently H or C₁-C₁₂ alkyl;    -   R⁸ and R⁹ are each independently unsubstituted C₁-C₁₂ alkyl; or        R⁸ and R⁹, together with the nitrogen atom to which they are        attached, form a 5, 6 or 7-membered heterocyclic ring comprising        one nitrogen atom;    -   a and d are each independently an integer from 0 to 24;    -   b and c are each independently an integer from 1 to 24;    -   e is 1 or 2; and    -   x is 0, 1 or 2.

In some embodiments of Formula I, L¹ and L² are independently —O(C═O)—or —(C═O)O—.

In certain embodiments of Formula I, at least one of R^(1a), R^(2a),R^(3a) or R^(4a) is C₁-C₁₂ alkyl, or at least one of L¹ or L² is—O(C═O)— or —(C═O)O—. In other embodiments, R^(1a) and R^(1b) are notisopropyl when a is 6 or n-butyl when a is 8.

In still further embodiments of Formula I, at least one of R^(1a),R^(2a), R^(3a) or R^(4a) is C₁-C₁₂ alkyl, or at least one of L¹ or L² is—O(C═O)— or —(C═O)O—; and

-   -   R^(1a) and R^(1b) are not isopropyl when a is 6 or n-butyl when        a is 8.

In other embodiments of Formula I, R⁸ and R⁹ are each independentlyunsubstituted C₁-C₁₂ alkyl; or R⁸ and R⁹, together with the nitrogenatom to which they are attached, form a 5, 6 or 7-membered heterocyclicring comprising one nitrogen atom;

In certain embodiments of Formula I, any one of L¹ or L² may be —O(C═O)—or a carbon-carbon double bond. L¹ and L² may each be —O(C═O)— or mayeach be a carbon-carbon double bond.

In some embodiments of Formula I, one of L¹ or L² is —O(C═O)—. In otherembodiments, both L¹ and L² are —O(C═O)—.

In some embodiments of Formula I, one of L¹ or L² is —(C═O)O—. In otherembodiments, both L¹ and L² are —(C═O)O—.

In some other embodiments of Formula I, one of L¹ or L² is acarbon-carbon double bond. In other embodiments, both L¹ and L² are acarbon-carbon double bond.

In still other embodiments of Formula I, one of L¹ or L² is —O(C═O)— andthe other of L¹ or L² is —(C═O)O—. In more embodiments, one of L¹ or L²is —O(C═O)— and the other of L¹ or L² is a carbon-carbon double bond. Inyet more embodiments, one of L¹ or L² is —(C═O)O— and the other of L¹ orL² is a carbon-carbon double bond.

It is understood that “carbon-carbon” double bond, as used throughoutthe specification, refers to one of the following structures:

wherein R^(a) and R^(b) are, at each occurrence, independently H or asubstituent. For example, in some embodiments R^(a) and R^(b) are, ateach occurrence, independently H, C₁-C₁₂ alkyl or cycloalkyl, forexample H or C₁-C₁₂ alkyl.

In other embodiments, the lipid compounds of Formula I have thefollowing Formula (Ia):

In other embodiments, the lipid compounds of Formula I have thefollowing Formula (Ib):

In yet other embodiments, the lipid compounds of Formula I have thefollowing Formula (Ic):

In certain embodiments of the lipid compound of Formula I, a, b, c and dare each independently an integer from 2 to 12 or an integer from 4 to12. In other embodiments, a, b, c and d are each independently aninteger from 8 to 12 or 5 to 9. In some certain embodiments, a is 0. Insome embodiments, a is 1. In other embodiments, a is 2. In moreembodiments, a is 3. In yet other embodiments, a is 4. In someembodiments, a is 5. In other embodiments, a is 6. In more embodiments,a is 7. In yet other embodiments, a is 8. In some embodiments, a is 9.In other embodiments, a is 10. In more embodiments, a is 11. In yetother embodiments, a is 12. In some embodiments, a is 13. In otherembodiments, a is 14. In more embodiments, a is 15. In yet otherembodiments, a is 16.

In some other embodiments of Formula I, b is 1. In other embodiments, bis 2. In more embodiments, b is 3. In yet other embodiments, b is 4. Insome embodiments, b is 5. In other embodiments, b is 6. In moreembodiments, b is 7. In yet other embodiments, b is 8. In someembodiments, b is 9. In other embodiments, b is 10. In more embodiments,b is 11. In yet other embodiments, b is 12. In some embodiments, b is13. In other embodiments, b is 14. In more embodiments, b is 15. In yetother embodiments, b is 16.

In some more embodiments of Formula I, c is 1. In other embodiments, cis 2. In more embodiments, c is 3. In yet other embodiments, c is 4. Insome embodiments, c is 5. In other embodiments, c is 6. In moreembodiments, c is 7. In yet other embodiments, c is 8. In someembodiments, c is 9. In other embodiments, c is 10. In more embodiments,c is 11. In yet other embodiments, c is 12. In some embodiments, c is13. In other embodiments, c is 14. In more embodiments, c is 15. In yetother embodiments, c is 16.

In some certain other embodiments of Formula I, d is 0. In someembodiments, d is 1. In other embodiments, d is 2. In more embodiments,d is 3. In yet other embodiments, d is 4. In some embodiments, d is 5.In other embodiments, d is 6. In more embodiments, d is 7. In yet otherembodiments, d is 8. In some embodiments, d is 9. In other embodiments,d is 10. In more embodiments, d is 11. In yet other embodiments, d is12. In some embodiments, d is 13. In other embodiments, d is 14. In moreembodiments, d is 15. In yet other embodiments, d is 16.

In some other various embodiments of Formula I, a and d are the same. Insome other embodiments, b and c are the same. In some other specificembodiments, a and d are the same and b and c are the same.

The sum of a and b and the sum of c and d in Formula I are factors whichmay be varied to obtain a lipid of formula I having the desiredproperties. In one embodiment, a and b are chosen such that their sum isan integer ranging from 14 to 24. In other embodiments, c and d arechosen such that their sum is an integer ranging from 14 to 24. Infurther embodiment, the sum of a and b and the sum of c and d are thesame. For example, in some embodiments the sum of a and b and the sum ofc and d are both the same integer which may range from 14 to 24. Instill more embodiments, a. b, c and d are selected such the sum of a andb and the sum of c and d is 12 or greater.

In some embodiments of Formula I, e is 1. In other embodiments, e is 2.

The substituents at R^(1a), R^(2a), R^(3a) and R^(4a) of Formula I arenot particularly limited. In certain embodiments R^(1a), R^(2a), R^(3a)and R^(4a) are H at each occurrence. In certain other embodiments atleast one of R^(1a), R^(2a), R^(3a) and R^(4a) is C₁-C₁₂ alkyl. Incertain other embodiments at least one of R^(1a), R^(2a), R^(3a) andR^(4a) is C₁-C₈ alkyl. In certain other embodiments at least one ofR^(1a), R^(2a), R^(3a) and R^(4a) is C₁-C₆ alkyl. In some of theforegoing embodiments, the C₁-C₈ alkyl is methyl, ethyl, n-propyl,iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

In certain embodiments of Formula I, R^(1a), R^(1b), R^(4a) and R^(4b)are C₁-C₁₂, alkyl at each occurrence.

In further embodiments of Formula I, at least one of R^(1b), R^(2b),R^(3b) and R^(4b) is H or R^(1b), R^(2b), R^(3b) and R^(4b) are H ateach occurrence.

In certain embodiments of Formula I, R^(1b) together with the carbonatom to which it is bound is taken together with an adjacent R^(1b) andthe carbon atom to which it is bound to form a carbon-carbon doublebond. In other embodiments of the foregoing R^(4b) together with thecarbon atom to which it is bound is taken together with an adjacentR^(4b) and the carbon atom to which it is bound to form a carbon-carbondouble bond.

The substituents at R⁵ and R⁶ of Formula I are not particularly limitedin the foregoing embodiments. In certain embodiments one or both of R⁵or R⁶ is methyl. In certain other embodiments one or both of R⁵ or R⁶ iscycloalkyl for example cyclohexyl. In these embodiments the cycloalkylmay be substituted or not substituted. In certain other embodiments thecycloalkyl is substituted with C₁-C₁₂ alkyl, for example tert-butyl.

The substituents at R⁷ are not particularly limited in the foregoingembodiments of Formula I. In certain embodiments at least one R⁷ is H.In some other embodiments, R⁷ is H at each occurrence. In certain otherembodiments R⁷ is C₁-C₁₂ alkyl.

In certain other of the foregoing embodiments of Formula I, one of R⁸ orR⁹ is methyl. In other embodiments, both R⁸ and R⁹ are methyl.

In some different embodiments of Formula I, R⁸ and R⁹, together with thenitrogen atom to which they are attached, form a 5, 6 or 7-memberedheterocyclic ring. In some embodiments of the foregoing, R⁸ and R⁹,together with the nitrogen atom to which they are attached, form a5-membered heterocyclic ring, for example a pyrrolidinyl ring.

In some embodiments of Embodiment 3, the first and second cationiclipids are each, independently selected from a lipid of Formula I.

In various different embodiments, the lipid of Formula I has one of thestructures set forth in Table 1 below.

TABLE 1 Representative Lipids of Formula I No. Structure pKa I-1

— I-2

5.64 I-3

7.15 I-4

6.43 I-5

6.28 I-6

6.12 I-7

— I-8

— I-9

— I-10

— I-11

6.36 I-12

— I-13

6.51 I-14

— I-15

6.30 I-16

6.63 I-17

— I-18

— I-19

6.72 I-20

6.44 I-21

6.28 I-22

6.53 I-23

6.24 I-24

6.28 I-25

6.20 I-33

6.27 I-34

— I-35

6.21 I-36

— I-37

— I-38

6.24 I-39

5.82 I-40

6.38 I-41

5.91

In some embodiments, the cationic lipid of Embodiments 1, 2, 3, 4 or 5has a structure of Formula II:

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomerthereof, wherein:

-   -   one of L¹ or L² is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)_(x)—,        —S—S—, —C(═O)S—, SC(═O)—, —NR^(a)C(═O)—, —C(═O)NR^(a)—,        NR^(a)C(═O)NR^(a)—, —OC(═O)NR^(a)— or —NR^(a)C(═O)O—, and the        other of L¹ or L² is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—,        —S(O)_(x)—, —S—S—, —C(═O)S—, SC(═O)—, —NR^(a)C(═O)—,        —C(═O)NR^(a)—, NR^(a)C(═O)NR^(a)—, —OC(═O)NR^(a)— or        —NR^(a)C(═O)O— or a direct bond;    -   G¹ is C₁-C₂ alkylene, —(C═O)—, —O(C═O)—, —SC(═O)—, —NR^(a)C(═O)—        or a direct bond;    -   G² is —C(═O)—, —(C═O)O—, —C(═O)S—, —C(═O)NR^(a)— or a direct        bond;    -   G³ is C₁-C₆ alkylene;    -   R^(a) is H or C₁-C₁₂ alkyl;    -   R^(1a) and R^(1b) are, at each occurrence, independently        either: (a) H or C₁-C₁₂ alkyl; or (b) R^(1a) is H or C₁-C₁₂        alkyl, and R^(1b) together with the carbon atom to which it is        bound is taken together with an adjacent R^(1b) and the carbon        atom to which it is bound to form a carbon-carbon double bond;    -   R^(2a) and R^(2b) are, at each occurrence, independently        either: (a) H or C₁-C₁₂ alkyl; or (b) R^(2a) is H or C₁-C₁₂        alkyl, and R^(2b) together with the carbon atom to which it is        bound is taken together with an adjacent R^(2b) and the carbon        atom to which it is bound to form a carbon-carbon double bond;    -   R^(3a) and R^(3b) are, at each occurrence, independently either        (a): H or C₁-C₁₂ alkyl; or (b) R^(3a) is H or C₁-C₁₂ alkyl, and        R^(3b) together with the carbon atom to which it is bound is        taken together with an adjacent R^(3b) and the carbon atom to        which it is bound to form a carbon-carbon double bond;    -   R^(4a) and R^(4b) are, at each occurrence, independently        either: (a) H or C₁-C₁₂ alkyl; or (b) R^(4a) is H or C₁-C₁₂        alkyl, and R^(4b) together with the carbon atom to which it is        bound is taken together with an adjacent R^(4b) and the carbon        atom to which it is bound to form a carbon-carbon double bond;    -   R⁵ and R⁶ are each independently H or methyl;    -   R⁷ is C₄-C₂₀ alkyl;    -   R⁸ and R⁹ are each independently C₁-C₁₂ alkyl; or R⁸ and R⁹,        together with the nitrogen atom to which they are attached, form        a 5, 6 or 7-membered heterocyclic ring;    -   a, b, c and d are each independently an integer from 1 to 24;        and    -   x is 0, 1 or 2.

In some embodiments of Formula (II), L¹ and L² are each independently—O(C═O)—, —(C═O)O— or a direct bond. In other embodiments, G¹ and G² areeach independently —(C═O)— or a direct bond. In some differentembodiments, L¹ and L² are each independently —O(C═O)—, —(C═O)O— or adirect bond; and G¹ and G² are each independently —(C═O)— or a directbond.

In some different embodiments of Formula (II), L¹ and L² are eachindependently —C(═O)—, —O—, —S(O)_(x)—, —S—S—, —C(═O)S—, —SC(═O)—,—NR^(a)—, —NR^(a)C(═O)—, —C(═O)NR^(a)—, NR^(a)C(═O)NR^(a),OC(═O)NR^(a)—, —NR^(a)C(═O)O—, —NR^(a)S(O)_(x)NR^(a)—, —NR^(a)S(O)_(x)—or —S(O)_(x)NR^(a)—.

In other of the foregoing embodiments of Formula (II), the lipidcompound has one of the following Formulae (IIA) or (IIB):

In some embodiments of Formula (II), the lipid compound has Formula(IIA). In other embodiments, the lipid compound has Formula (IIB).

In any of the foregoing embodiments of Formula (II), one of L¹ or L² is—O(C═O)—. For example, in some embodiments each of L¹ and L² are—O(C═O)—.

In some different embodiments of Formula (II), one of L¹ or L² is—(C═O)O—. For example, in some embodiments each of L¹ and L² is—(C═O)O—.

In different embodiments of Formula (II), one of L¹ or L² is a directbond. As used herein, a “direct bond” means the group (e.g., L¹ or L²)is absent. For example, in some embodiments each of L¹ and L² is adirect bond.

In other different embodiments of Formula (II), for at least oneoccurrence of R^(1a) and R^(1b), R^(1a) is H or C₁-C₁₂ alkyl, and R^(1b)together with the carbon atom to which it is bound is taken togetherwith an adjacent R^(1b) and the carbon atom to which it is bound to forma carbon-carbon double bond.

In still other different embodiments of Formula (II), for at least oneoccurrence of R^(4a) and R^(4b), R^(4a) is H or C₁-C₁₂ alkyl, and R^(4b)together with the carbon atom to which it is bound is taken togetherwith an adjacent R^(4b) and the carbon atom to which it is bound to forma carbon-carbon double bond.

In more embodiments of Formula (II), for at least one occurrence ofR^(2a) and R^(2b), R^(2a) is H or C₁-C₁₂ alkyl, and R^(2b) together withthe carbon atom to which it is bound is taken together with an adjacentR^(2b) and the carbon atom to which it is bound to form a carbon-carbondouble bond.

In other different embodiments of Formula (II), for at least oneoccurrence of R^(3a) and R^(3b), R^(3a) is H or C₁-C₁₂ alkyl, and R^(3b)together with the carbon atom to which it is bound is taken togetherwith an adjacent R^(3b) and the carbon atom to which it is bound to forma carbon-carbon double bond.

In various other embodiments of Formula (II), the lipid compound has oneof the following Formulae (IIC) or (HD):

wherein e, f, g and h are each independently an integer from 1 to 12.

In some embodiments of Formula (II), the lipid compound has Formula(IIC). In other embodiments, the lipid compound has Formula (IID).

In various embodiments of Formulae (IIC) or (IID), e, f, g and h areeach independently an integer from 4 to 10.

In certain embodiments of Formula (II), a, b, c and d are eachindependently an integer from 2 to 12 or an integer from 4 to 12. Inother embodiments, a, b, c and d are each independently an integer from8 to 12 or 5 to 9. In some certain embodiments, a is 0. In someembodiments, a is 1. In other embodiments, a is 2. In more embodiments,a is 3. In yet other embodiments, a is 4. In some embodiments, a is 5.In other embodiments, a is 6. In more embodiments, a is 7. In yet otherembodiments, a is 8. In some embodiments, a is 9. In other embodiments,a is 10. In more embodiments, a is 11. In yet other embodiments, a is12. In some embodiments, a is 13. In other embodiments, a is 14. In moreembodiments, a is 15. In yet other embodiments, a is 16.

In some embodiments of Formula (II), b is 1. In other embodiments, b is2. In more embodiments, b is 3. In yet other embodiments, b is 4. Insome embodiments, b is 5. In other embodiments, b is 6. In moreembodiments, b is 7. In yet other embodiments, b is 8. In someembodiments, b is 9. In other embodiments, b is 10. In more embodiments,b is 11. In yet other embodiments, b is 12. In some embodiments, b is13. In other embodiments, b is 14. In more embodiments, b is 15. In yetother embodiments, b is 16.

In some embodiments of Formula (II), c is 1. In other embodiments, c is2. In more embodiments, c is 3. In yet other embodiments, c is 4. Insome embodiments, c is 5. In other embodiments, c is 6. In moreembodiments, c is 7. In yet other embodiments, c is 8. In someembodiments, c is 9. In other embodiments, c is 10. In more embodiments,c is 11. In yet other embodiments, c is 12. In some embodiments, c is13. In other embodiments, c is 14. In more embodiments, c is 15. In yetother embodiments, c is 16.

In some certain embodiments of Formula (II), d is 0. In someembodiments, d is 1. In other embodiments, d is 2. In more embodiments,d is 3. In yet other embodiments, d is 4. In some embodiments, d is 5.In other embodiments, d is 6. In more embodiments, d is 7. In yet otherembodiments, d is 8. In some embodiments, d is 9. In other embodiments,d is 10. In more embodiments, d is 11. In yet other embodiments, d is12. In some embodiments, d is 13. In other embodiments, d is 14. In moreembodiments, d is 15. In yet other embodiments, d is 16.

In some embodiments of Formula (II), e is 1. In other embodiments, e is2. In more embodiments, e is 3. In yet other embodiments, e is 4. Insome embodiments, e is 5. In other embodiments, e is 6. In moreembodiments, e is 7. In yet other embodiments, e is 8. In someembodiments, e is 9. In other embodiments, e is 10. In more embodiments,e is 11. In yet other embodiments, e is 12.

In some embodiments of Formula (II), f is 1. In other embodiments, f is2. In more embodiments, f is 3. In yet other embodiments, f is 4. Insome embodiments, f is 5. In other embodiments, f is 6. In moreembodiments, f is 7. In yet other embodiments, f is 8. In someembodiments, f is 9. In other embodiments, f is 10. In more embodiments,f is 11. In yet other embodiments, f is 12.

In some embodiments of Formula (II), g is 1. In other embodiments, g is2. In more embodiments, g is 3. In yet other embodiments, g is 4. Insome embodiments, g is 5. In other embodiments, g is 6. In moreembodiments, g is 7. In yet other embodiments, g is 8. In someembodiments, g is 9. In other embodiments, g is 10. In more embodiments,g is 11. In yet other embodiments, g is 12.

In some embodiments of Formula (II), his 1. In other embodiments, e is2. In more embodiments, h is 3. In yet other embodiments, h is 4. Insome embodiments, e is 5. In other embodiments, h is 6. In moreembodiments, h is 7. In yet other embodiments, h is 8. In someembodiments, h is 9. In other embodiments, h is 10. In more embodiments,his 11. In yet other embodiments, his 12.

In some other various embodiments of Formula (II), a and d are the same.In some other embodiments, b and c are the same. In some other specificembodiments and a and d are the same and b and c are the same.

The sum of a and b and the sum of c and d of Formula (II) are factorswhich may be varied to obtain a lipid having the desired properties. Inone embodiment, a and b are chosen such that their sum is an integerranging from 14 to 24. In other embodiments, c and d are chosen suchthat their sum is an integer ranging from 14 to 24. In furtherembodiment, the sum of a and b and the sum of c and d are the same. Forexample, in some embodiments the sum of a and b and the sum of c and dare both the same integer which may range from 14 to 24. In still moreembodiments, a. b, c and d are selected such that the sum of a and b andthe sum of c and d is 12 or greater.

The substituents at R^(1a), R^(2a), R^(3a) and R^(4a) of Formula (II)are not particularly limited. In some embodiments, at least one ofR^(1a), R^(2a), R^(3a) and R^(4a) is H. In certain embodiments R^(1a),R^(2a), R^(3a) and R^(4a) are H at each occurrence. In certain otherembodiments at least one of R^(1a), R^(2a), R^(3a) and R^(4a) is C₁-C₁₂alkyl. In certain other embodiments at least one of R^(1a), R^(2a),R^(3a) and R^(4a) is C₁-C₈ alkyl. In certain other embodiments at leastone of R^(1a), R^(2a), R^(3a) and R^(4a) is C₁-C₆ alkyl. In some of theforegoing embodiments, the C₁-C₈ alkyl is methyl, ethyl, n-propyl,iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

In certain embodiments of Formula (II), R^(1a), R^(1b), R^(4a) andR^(4b) are C₁-C₁₂, alkyl at each occurrence.

In further embodiments of Formula (II), at least one of R^(1b), R^(2b),R^(3b) and R^(4b) is H or R^(1b), R^(2b), R^(3b) and R^(4b) are H ateach occurrence.

In certain embodiments of Formula (II), R^(1b) together with the carbonatom to which it is bound is taken together with an adjacent R^(1b) andthe carbon atom to which it is bound to form a carbon-carbon doublebond. In other embodiments of the foregoing R^(4b) together with thecarbon atom to which it is bound is taken together with an adjacentR^(4b) and the carbon atom to which it is bound to form a carbon-carbondouble bond.

The substituents at R⁵ and R⁶ of Formula (II) are not particularlylimited in the foregoing embodiments. In certain embodiments one of R⁵or R⁶ is methyl. In other embodiments each of R⁵ or R⁶ is methyl.

The substituents at R⁷ of Formula (II) are not particularly limited inthe foregoing embodiments. In certain embodiments R⁷ is C₆-C₁₆ alkyl. Insome other embodiments, R⁷ is C₆-C₉ alkyl. In some of these embodiments,R⁷ is substituted with —(C═O)OR^(b), —O(C═O)R^(b), —C(═O)R^(b), —OR^(b),—S(O)_(x)R^(b), —S—SR^(b), —C(═O)SR^(b), —SC(═O)R^(b), —NR^(a)R^(b),—NR^(a)C(═O)R^(b), —C(═O)NR^(a)R^(b), —NR^(a)C(═O)NR^(a)R^(b),—OC(═O)NR^(a)R^(b), —NR^(a)C(═O)OR^(b), —NR^(a)S(O)_(x)NR^(a)R^(b),—NR^(a)S(O)_(x)R^(b) or —S(O)_(x)NR^(a)R^(b), wherein: R^(a) is H orC₁-C₁₂ alkyl; R^(b) is C₁-C₁₅ alkyl; and x is 0, 1 or 2. For example, insome embodiments R⁷ is substituted with —(C═O)OR^(b) or —O(C═O)R^(b).

In some of the foregoing embodiments of Formula (II), R^(b) is branchedC₁-C₁₆ alkyl. For example, in some embodiments R^(b) has one of thefollowing structures:

In certain other of the foregoing embodiments of Formula (II), one of R⁸or R⁹ is methyl. In other embodiments, both R⁸ and R⁹ are methyl.

In some different embodiments of Formula (II), R⁸ and R⁹, together withthe nitrogen atom to which they are attached, form a 5, 6 or 7-memberedheterocyclic ring. In some embodiments of the foregoing, R⁸ and R⁹,together with the nitrogen atom to which they are attached, form a5-membered heterocyclic ring, for example a pyrrolidinyl ring. In somedifferent embodiments of the foregoing, R⁸ and R⁹, together with thenitrogen atom to which they are attached, form a 6-membered heterocyclicring, for example a piperazinyl ring.

In certain embodiments of Embodiment 3, the first and second cationiclipids are each, independently selected from a lipid of Formula II.

In still other embodiments of the foregoing lipids of Formula (II), G³is C₂-C₄ alkylene, for example C₃ alkylene. In various differentembodiments, the lipid compound has one of the structures set forth inTable 2 below

TABLE 2 Representative Lipids of Formula (II) No. Structure pKa II-1

5.64 II-2

— II-3

— II-4

— II-5

6.27 II-6

6.14 II-7

5.93 II-8

5.35 II-9

6.27 II-10

6.16 II-11

6.13 II-12

6.21 II-13

6.22 II-14

6.33 II-15

6.32 II-16

6.37 II-17

6.27 II-18

— II-19

— II-20

— II-21

— II-22

— II-23

— II-24

6.14 II-25

— II-26

— II-27

— II-28

— II-29

— II-30

— II-31

— II-32

— II-33

— II-34

— II-35

5.97 II-36

6.13 II-37

5.61 II-38

6.45 II-39

6.45 II-40

6.57 II-41

— II-42

— II-43

— II-44

— II-45

— II-46

—

In some other embodiments, the cationic lipid of Embodiments 1, 2, 3, 4or 5 has a structure of Formula III:

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof,wherein:

-   -   one of L¹ or L² is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)_(x),        —S—S—, —C(═O)S—, SC(═O)—, —NR^(a)C(═O)—, —C(═O)NR^(a)—,        NR^(a)C(═O)NR^(a)—, —OC(═O)NR^(a)— or —NR^(a)C(═O)O—, and the        other of L¹ or L² is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—,        —S(O)_(x)—, —S—S—, —C(═O)S—, SC(═O)—, —NR^(a)C(═O)—,        —C(═O)NR^(a)—, NR^(a)C(═O)NR^(a)—, —OC(═O)NR^(a)— or        —NR^(a)C(═O)O— or a direct bond;    -   G¹ and G² are each independently unsubstituted C₁-C₁₂ alkylene        or C₁-C₁₂ alkenylene;    -   G³ is C₁-C₂₄ alkylene, C₁-C₂₄ alkenylene, C₃-C₈ cycloalkylene,        C₃-C₈ cycloalkenylene;    -   R^(a) is H or C₁-C₁₂ alkyl;    -   R¹ and R² are each independently C₆-C₂₄ alkyl or C₆-C₂₄ alkenyl;    -   R³ is H, OR⁵, CN, —C(═O)OR⁴, —OC(═O)R⁴ or —NR⁵C(═O)R⁴;    -   R⁴ is C₁-C₁₂ alkyl;    -   R⁵ is H or C₁-C₆ alkyl; and    -   x is 0, 1 or 2.

In some of the foregoing embodiments of Formula (III), the lipid has oneof the following Formulae (IIIA) or (TIM):

wherein:

-   -   A is a 3 to 8-membered cycloalkyl or cycloalkylene ring;    -   R⁶ is, at each occurrence, independently H, OH or C₁-C₂₄ alkyl;    -   n is an integer ranging from 1 to 15.

In some of the foregoing embodiments of Formula (III), the lipid hasFormula (IIIA), and in other embodiments, the lipid has Formula (IIIB).

In other embodiments of Formula (III), the lipid has one of thefollowing Formulae (IIIC) or (IIID):

wherein y and z are each independently integers ranging from 1 to 12.

In any of the foregoing embodiments of Formula (III), one of L¹ or L² is—O(C═O)—. For example, in some embodiments each of L¹ and L² are—O(C═O)—. In some different embodiments of any of the foregoing, L¹ andL² are each independently —(C═O)O— or —O(C═O)— For example, in someembodiments each of L¹ and L² is —(C═O)O—.

In some different embodiments of Formula (III), the lipid has one of thefollowing Formulae (IIIE) or (IIIF):

In some of the foregoing embodiments of Formula (III), the lipid has oneof the following Formulae (IIIG), (IIIH), (IIII), or (IIIJ):

In some of the foregoing embodiments of Formula (III), n is an integerranging from 2 to 12, for example from 2 to 8 or from 2 to 4. Forexample, in some embodiments, n is 3, 4, 5 or 6. In some embodiments, nis 3. In some embodiments, n is 4. In some embodiments, n is 5. In someembodiments, n is 6.

In some other of the foregoing embodiments of Formula (III), y and z areeach independently an integer ranging from 2 to 10. For example, in someembodiments, y and z are each independently an integer ranging from 4 to9 or from 4 to 6.

In some of the foregoing embodiments of Formula (III), R⁶ is H. In otherof the foregoing embodiments, R⁶ is C₁-C₂₄ alkyl. In other embodiments,R⁶ is OH.

In some embodiments of Formula (III), G³ is unsubstituted. In otherembodiments, G3 is substituted. In various different embodiments, G³ islinear C₁-C₂₄ alkylene or linear C₁-C₂₄ alkenylene.

In some other foregoing embodiments of Formula (III), R¹ or R², or both,is C₆-C₂₄ alkenyl. For example, in some embodiments, R¹ and R² each,independently have the following structure:

wherein:

-   -   R^(7a) and R^(7b) are, at each occurrence, independently H or        C₁-C₁₂ alkyl; and    -   a is an integer from 2 to 12,

wherein R^(7a), R^(7b) and a are each selected such that R¹ and R² eachindependently comprise from 6 to 20 carbon atoms. For example, in someembodiments a is an integer ranging from 5 to 9 or from 8 to 12.

In some of the foregoing embodiments of Formula (III), at least oneoccurrence of R^(7a) is H. For example, in some embodiments, R^(7a) is Hat each occurrence. In other different embodiments of the foregoing, atleast one occurrence of R^(7b) is C₁-C₈ alkyl. For example, in someembodiments, C₁-C₈ alkyl is methyl, ethyl, n-propyl, iso-propyl,n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

In different embodiments of Formula (III), R¹ or R², or both, has one ofthe following structures:

In some of the foregoing embodiments of Formula (III), R³ is OH, CN,—C(═O)OR⁴, —OC(═O)R⁴ or —NHC(═O)R⁴. In some embodiments, R⁴ is methyl orethyl.

In some specific embodiments of Embodiment 3, the first and secondcationic lipids are each, independently selected from a lipid of FormulaIII.

In various different embodiments, a cationic lipid of any one of thedisclosed embodiments (e.g., the cationic lipid, the first cationiclipid, the second cationic lipid) of Formula (III) has one of thestructures set forth in Table 3 below.

TABLE 3 Representative Compounds of Formula (III) No. Structure pKaIII-1

5.89 III-2

6.05 III-3

6.09 III-4

5.60 III-5

5.59 III-6

5.42 III-7

6.11 III-8

5.84 III-9

— III-10

— III-11

— III-12

— III-13

— III-14

— III-15

6.14 III-16

6.31 III-17

6.28 III-18

— III-19

— III-20

6.36 III-21

— III-22

6.10 III-23

5.98 III-24

— III-25

6.22 III-26

5.84 III-27

5.77 III-28

— III-29

— III-30

6.09 III-31

— III-32

— III-33

— III-34

— III-35

— III-36

— III-37

— III-38

— III-39

— III-40

— III-41

— III-42

— III-43

— III-44

— III-45

— III-46

— III-47

— III-48

— III-49

—

In one embodiment, the cationic lipid of any one of Embodiments 1, 2, 3,4 or 5 has a structure of Formula (IV):

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof,wherein:

-   -   one of G¹ or G² is, at each occurrence, —O(C═O)—, —(C═O)O—,        —C(═O)—, —O—, —S(O)_(y)—, —S—S—, —C(═O)S—, SC(═O)—,        —N(R^(a))C(═O)—, —C(═O)N(R^(a))—, —N(R^(a))C(═O)N(R^(a))—,        —OC(═O)N(R^(a))— or —N(R^(a))C(═O)O—, and the other of G¹ or G²        is, at each occurrence, —O(C═O)—, —(C═O)O—, —C(═O)—, —O—,        —S(O)_(y)—, —S—S—, —C(═O)S—, —SC(═O)—, —N(R^(a))C(═O)—,        —C(═O)N(R^(a))—, —N(R^(a))C(═O)N(R^(a))—, —OC(═O)N(R^(a))— or        —N(R^(a))C(═O)O— or a direct bond;    -   L is, at each occurrence, ˜O(C═O)—, wherein ˜ represents a        covalent bond to X;    -   X is CR^(a);    -   Z is alkyl, cycloalkyl or a monovalent moiety comprising at        least one polar functional group when n is 1; or Z is alkylene,        cycloalkylene or a polyvalent moiety comprising at least one        polar functional group when n is greater than 1;    -   R^(a) is, at each occurrence, independently H, C₁-C₁₂ alkyl,        C₁-C₁₂ hydroxylalkyl, aminoalkyl, alkylaminylalkyl, C₁-C₁₂        alkoxyalkyl, C₁-C₁₂ alkoxycarbonyl, C₁-C₁₂ alkylcarbonyloxy,        C₁-C₁₂ alkylcarbonyloxyalkyl or C₁-C₁₂ alkylcarbonyl;    -   R is, at each occurrence, independently either: (a) H or C₁-C₁₂        alkyl; or (b) R together with the carbon atom to which it is        bound is taken together with an adjacent R and the carbon atom        to which it is bound to form a carbon-carbon double bond;    -   R¹ and R² have, at each occurrence, the following structure,        respectively:

-   -   a¹ and a² are, at each occurrence, independently an integer from        3 to 12;    -   b¹ and b² are, at each occurrence, independently 0 or 1;    -   c¹ and c² are, at each occurrence, independently an integer from        5 to 10;    -   d¹ and d² are, at each occurrence, independently an integer from        5 to 10;    -   y is, at each occurrence, independently an integer from 0 to 2;        and    -   n is an integer from 1 to 6,    -   wherein each alkyl, alkylene, hydroxylalkyl, aminoalkyl,        alkylaminylalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy,        alkylcarbonyloxyalkyl and alkylcarbonyl is optionally        substituted with one or more substituent.

In some embodiments of Formula (IV), G¹ and G² are each independentlyO(C═O)— or —(C═O)O—.

In other embodiments of Formula (IV), X is CH.

In different embodiments of Formula (IV), the sum of a¹+b¹+c¹ or the sumof a²+b²+c² is an integer from 12 to 26.

In still other embodiments of Formula (IV), a¹ and a² are independentlyan integer from 3 to 10. For example, in some embodiments a¹ and a² areindependently an integer from 4 to 9.

In various embodiments of Formula (IV), b¹ and b² are 0. In differentembodiments, b¹ and b² are 1.

In more embodiments of Formula (IV), c¹, c², d¹ and d² are independentlyan integer from 6 to 8.

In other embodiments of Formula (IV), c¹ and c² are, at each occurrence,independently an integer from 6 to 10, and d¹ and d² are, at eachoccurrence, independently an integer from 6 to 10.

In other embodiments of Formula (IV), c¹ and c² are, at each occurrence,independently an integer from 5 to 9, and d¹ and d² are, at eachoccurrence, independently an integer from 5 to 9.

In more embodiments of Formula (IV), Z is alkyl, cycloalkyl or amonovalent moiety comprising at least one polar functional group when nis 1. In other embodiments, Z is alkyl.

In various embodiments of the foregoing Formula (IV), R is, at eachoccurrence, independently either: (a) H or methyl; or (b) R togetherwith the carbon atom to which it is bound is taken together with anadjacent R and the carbon atom to which it is bound to form acarbon-carbon double bond. In certain embodiments, each R is H. In otherembodiments at least one R together with the carbon atom to which it isbound is taken together with an adjacent R and the carbon atom to whichit is bound to form a carbon-carbon double bond.

In other embodiments of the compound of Formula (IV), R¹ and R²independently have one of the following structures:

In certain embodiments of Formula (IV), the compound has one of thefollowing structures:

In still different embodiments the cationic lipid of Embodiments 1, 2,3, 4 or 5 has the structure of Formula (V):

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof,wherein:

-   -   one of G¹ or G² is, at each occurrence, —O(C═O)—, —(C═O)O—,        —C(═O)—, —O—, —S(O)₅—, —S—S—, —C(═O)S—, SC(═O)—,        —N(R^(a))C(═O)—, —C(═O)N(R^(a))—, —N(R^(a))C(═O)N(R^(a))—,        —OC(═O)N(R^(a))— or —N(R^(a))C(═O)O—, and the other of G¹ or G²        is, at each occurrence, —O(C═O)—, —(C═O)O—, —C(═O)—, —O—,        —S(O)_(y)—, —S—S—, —C(═O)S—, —SC(═O)—, —N(R^(a))C(═O)—,        —C(═O)N(R^(a))—, —N(R^(a))C(═O)N(R^(a))—, —OC(═O)N(R^(a))— or        —N(R^(a))C(═O)O— or a direct bond;    -   L is, at each occurrence, ˜O(C═O)—, wherein ˜ represents a        covalent bond to X,    -   X is CR^(a);    -   Z is alkyl, cycloalkyl or a monovalent moiety comprising at        least one polar functional group when n is 1; or Z is alkylene,        cycloalkylene or a polyvalent moiety comprising at least one        polar functional group when n is greater than 1;    -   R^(a) is, at each occurrence, independently H, C₁-C₁₂ alkyl,        C₁-C₁₂ hydroxylalkyl, C₁-C₁₂ aminoalkyl, C₁-C₁₂        alkylaminylalkyl, C₁-C₁₂ alkoxyalkyl, C₁-C₁₂ alkoxycarbonyl,        C₁-C₁₂ alkylcarbonyloxy, C₁-C₁₂ alkylcarbonyloxyalkyl or C₁-C₁₂        alkylcarbonyl;    -   R is, at each occurrence, independently either: (a) H or C₁-C₁₂        alkyl; or (b) R together with the carbon atom to which it is        bound is taken together with an adjacent R and the carbon atom        to which it is bound to form a carbon-carbon double bond;    -   R¹ and R² have, at each occurrence, the following structure,        respectively:

-   -   R′ is, at each occurrence, independently H or C₁-C₁₂ alkyl;    -   a¹ and a² are, at each occurrence, independently an integer from        3 to 12;    -   b¹ and b² are, at each occurrence, independently 0 or 1;    -   c¹ and c² are, at each occurrence, independently an integer from        2 to 12;    -   d¹ and d² are, at each occurrence, independently an integer from        2 to 12;    -   y is, at each occurrence, independently an integer from 0 to 2;        and    -   n is an integer from 1 to 6,    -   wherein a¹, a², c¹, c², d¹ and d² are selected such that the sum        of a¹+c¹+d¹ is an integer from 18 to 30, and the sum of a²+c²+d²        is an integer from 18 to 30, and wherein each alkyl, alkylene,        hydroxylalkyl, aminoalkyl, alkylaminylalkyl, alkoxyalkyl,        alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl and        alkylcarbonyl is optionally substituted with one or more        substituent.

In certain embodiments of Formula (V), G¹ and G² are each independently

-   -   O(C═O)— or —(C═O)O—.

In other embodiments of Formula (V), X is CH.

In some embodiments of Formula (V), the sum of a¹+c¹+d¹ is an integerfrom 20 to 30, and the sum of a²+c²+d² is an integer from 18 to 30. Inother embodiments, the sum of a¹+c¹+d¹ is an integer from 20 to 30, andthe sum of a²+c²+d² is an integer from 20 to 30. In more embodiments ofFormula (V), the sum of a¹+b¹+c¹ or the sum of a²+b²+c² is an integerfrom 12 to 26. In other embodiments, a¹, a², c¹, c², d¹ and d² areselected such that the sum of a¹+c¹+d¹ is an integer from 18 to 28, andthe sum of a²+c²+d² is an integer from 18 to 28,

In still other embodiments of Formula (V), a¹ and a² are independentlyan integer from 3 to 10, for example an integer from 4 to 9.

In yet other embodiments of Formula (V), b¹ and b² are 0. In differentembodiments b¹ and b² are 1.

In certain other embodiments of Formula (V), c¹, c², d¹ and d² areindependently an integer from 6 to 8.

In different other embodiments of Formula (V), Z is alkyl or amonovalent moiety comprising at least one polar functional group when nis 1; or Z is alkylene or a polyvalent moiety comprising at least onepolar functional group when n is greater than 1.

In more embodiments of Formula (V), Z is alkyl, cycloalkyl or amonovalent moiety comprising at least one polar functional group when nis 1. In other embodiments, Z is alkyl.

In other different embodiments of Formula (V), R is, at each occurrence,independently either: (a) H or methyl; or (b) R together with the carbonatom to which it is bound is taken together with an adjacent R and thecarbon atom to which it is bound to form a carbon-carbon double bond.For example in some embodiments each R is H. In other embodiments atleast one R together with the carbon atom to which it is bound is takentogether with an adjacent R and the carbon atom to which it is bound toform a carbon-carbon double bond.

In more embodiments, each R′ is H.

In certain embodiments of Formula (V), the sum of a¹+c¹+d¹ is an integerfrom 20 to 25, and the sum of a²+c²+d² is an integer from 20 to 25.

In other embodiments of Formula (V), R¹ and R² independently have one ofthe following structures:

In more embodiments of Formula (V), the compound has one of thefollowing structures:

In any of the foregoing embodiments of Formula (IV) or (V), n is 1. Inother of the foregoing embodiments of Formula (IV) or (V), n is greaterthan 1.

In more of any of the foregoing embodiments of Formula (IV) or (V), Z isa mono- or polyvalent moiety comprising at least one polar functionalgroup. In some embodiments, Z is a monovalent moiety comprising at leastone polar functional group. In other embodiments, Z is a polyvalentmoiety comprising at least one polar functional group.

In more of any of the foregoing embodiments of Formula (IV) or (V), thepolar functional group is a hydroxyl, alkoxy, ester, cyano, amide,amino, alkylaminyl, heterocyclyl or heteroaryl functional group.

In any of the foregoing embodiments of Formula (IV) or (V), Z ishydroxyl, hydroxylalkyl, alkoxyalkyl, amino, aminoalkyl, alkylaminyl,alkylaminylalkyl, heterocyclyl or heterocyclylalkyl.

In some other embodiments of Formula (IV) or (V), Z has the followingstructure:

wherein:

-   -   R⁵ and R⁶ are independently H or C₁-C₆ alkyl;    -   R⁷ and R⁸ are independently H or C₁-C₆ alkyl or R⁷ and R⁸,        together with the nitrogen atom to which they are attached, join        to form a 3-7 membered heterocyclic ring; and    -   x is an integer from 0 to 6.

In still different embodiments of Formula (IV) or (V), Z has thefollowing structure:

wherein:

-   -   R⁵ and R⁶ are independently H or C₁-C₆ alkyl;    -   R⁷ and R⁸ are independently H or C₁-C₆ alkyl or R⁷ and R⁸,        together with the nitrogen atom to which they are attached, join        to form a 3-7 membered heterocyclic ring; and    -   x is an integer from 0 to 6.

In still different embodiments of formula (IV) or (V), Z has thefollowing structure:

wherein:

-   -   R⁵ and R⁶ are independently H or C₁-C₆ alkyl;    -   R⁷ and R⁸ are independently H or C₁-C₆ alkyl or R⁷ and R⁸,        together with the nitrogen atom to which they are attached, join        to form a 3-7 membered heterocyclic ring; and    -   x is an integer from 0 to 6.

In some other embodiments of Formula (IV) or (V), Z is hydroxylalkyl,cyanoalkyl or an alkyl substituted with one or more ester or amidegroups.

For example, in any of the foregoing embodiments of Formula (IV) or (V),Z has one of the following structures:

In other embodiments of Formula (IV) or (V), Z-L has one of thefollowing structures:

In other embodiments, Z-L has one of the following structures:

In still other embodiments, X is CH and Z-L has one of the followingstructures:

In various different embodiments, a cationic lipid of any oneEmbodiments 1, 2, 3, 4 or 5 has one of the structures set forth in Table4 below.

TABLE 4 Representative Compounds of Formula (IV) or (V) No. StructureIV-1

IV-2

IV-3

In one embodiment, the cationic lipid is a compound having the followingstructure (VI):

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomerthereof, wherein:

-   -   L¹ and L² are each independently —O(C═O)—, —(C═O)O—, —C(═O)—,        —O—, —S(O)_(x)—, —S—S—, —C(═O)S—, —SC(═O)—, —NR^(a)C(═O)—,        —C(═O)NR^(a)—, —NR^(a)C(═O)NR^(a)—, —OC(═O)NR^(a)—,        —NR^(a)C(═O)O— or a direct bond;    -   G¹ is C₁-C₂ alkylene, —(C═O)—, —O(C═O)—, —SC(═O)—, —NR^(a)C(═O)—        or a direct bond;    -   G² is —C(═O)—, —(C═O)O—, —C(═O)S—, —C(═O)NR^(a)— or a direct        bond;    -   G³ is C₁-C₆ alkylene;    -   R^(a) is H or C₁-C₁₂ alkyl;    -   R^(1a) and R^(1b) are, at each occurrence, independently        either: (a) H or C₁-C₁₂ alkyl; or (b) R^(1a) is H or C₁-C₁₂        alkyl, and R^(1b) together with the carbon atom to which it is        bound is taken together with an adjacent R^(1b) and the carbon        atom to which it is bound to form a carbon-carbon double bond;    -   R^(2a) and R^(2b) are, at each occurrence, independently        either: (a) H or C₁-C₁₂ alkyl; or (b) R^(2a) is H or C₁-C₁₂        alkyl, and R^(2b) together with the carbon atom to which it is        bound is taken together with an adjacent R^(2b) and the carbon        atom to which it is bound to form a carbon-carbon double bond;    -   R^(3a) and R^(3b) are, at each occurrence, independently either        (a): H or C₁-C₁₂ alkyl; or (b) R^(3a) is H or C₁-C₁₂ alkyl, and        R^(3b) together with the carbon atom to which it is bound is        taken together with an adjacent R^(3b) and the carbon atom to        which it is bound to form a carbon-carbon double bond;    -   R^(4a) and R^(4b) are, at each occurrence, independently        either: (a) H or C₁-C₁₂ alkyl; or (b) R^(4a) is H or C₁-C₁₂        alkyl, and R^(4b) together with the carbon atom to which it is        bound is taken together with an adjacent R^(4b) and the carbon        atom to which it is bound to form a carbon-carbon double bond;    -   R⁵ and R⁶ are each independently H or methyl;    -   R⁷ is H or C₁-C₂₀ alkyl;    -   R⁸ is OH, —N(R⁹)(C═O)R¹⁰, —(C═O)NR⁹R¹⁰, —NR⁹R¹⁰, —(C═O)OR¹¹ or        —O(C═O)R¹¹, provided that G³ is C₄-C₆ alkylene when R⁸ is        —NR⁹R¹⁰,    -   R⁹ and R¹⁰ are each independently H or C₁-C₁₂ alkyl;    -   R¹¹ is aralkyl;    -   a, b, c and d are each independently an integer from 1 to 24;        and    -   x is 0, 1 or 2,        wherein each alkyl, alkylene and aralkyl is optionally        substituted.

In some embodiments of structure (VI), L¹ and L² are each independently—O(C═O)—, —(C═O)O— or a direct bond. In other embodiments, G¹ and G² areeach independently —(C═O)— or a direct bond. In some differentembodiments, L¹ and L² are each independently —O(C═O)—, —(C═O)O— or adirect bond; and G¹ and G² are each independently —(C═O)— or a directbond.

In some different embodiments of structure (VI), L¹ and L² are eachindependently —C(═O)—, —O—, —S(O)_(x), —S—S—, —C(═O)S—, —SC(═O)—,—NR^(a)—, —NR^(a)C(═O)—, —C(═O)NR^(a)—, —NR^(a)C(═O)NR^(a),—OC(═O)NR^(a)—, —NR^(a)C(═O)O—, —NR^(a)S(O)_(x)NR^(a)—, —NR^(a)S(O)_(x)—or —S(O)_(x)NR^(a)—.

In other of the foregoing embodiments of structure (VI), the compoundhas one of the following structures (VIA) or (VIB):

In some embodiments, the compound has structure (VIA). In otherembodiments, the compound has structure (VIB).

In any of the foregoing embodiments of structure (VI), one of L¹ or L²is —O(C═O)—. For example, in some embodiments each of L¹ and L² are—O(C═O)—.

In some different embodiments of any of the foregoing, one of L¹ or L²is —(C═O)O—. For example, in some embodiments each of L¹ and L² is—(C═O)O—.

In different embodiments of structure (VI), one of L¹ or L² is a directbond. As used herein, a “direct bond” means the group (e.g., L¹ or L²)is absent. For example, in some embodiments each of L¹ and L² is adirect bond.

In other different embodiments of the foregoing, for at least oneoccurrence of R^(1a) and R^(1b), R^(1a) is H or C₁-C₁₂ alkyl, and R^(1b)together with the carbon atom to which it is bound is taken togetherwith an adjacent R^(1b) and the carbon atom to which it is bound to forma carbon-carbon double bond.

In still other different embodiments of structure (VI), for at least oneoccurrence of R^(4a) and R_(4b), R^(4a) is H or C₁-C₁₂ alkyl, and R^(4b)together with the carbon atom to which it is bound is taken togetherwith an adjacent R^(4b) and the carbon atom to which it is bound to forma carbon-carbon double bond.

In more embodiments of structure (VI), for at least one occurrence ofR^(2a) and R_(2b), R^(2a) is H or C₁-C₁₂ alkyl, and R^(2b) together withthe carbon atom to which it is bound is taken together with an adjacentR^(2b) and the carbon atom to which it is bound to form a carbon-carbondouble bond.

In other different embodiments of any of the foregoing, for at least oneoccurrence of R^(3a) and R^(3b), R^(3a) is H or C₁-C₁₂ alkyl, and R^(3b)together with the carbon atom to which it is bound is taken togetherwith an adjacent R^(3b) and the carbon atom to which it is bound to forma carbon-carbon double bond.

It is understood that “carbon-carbon” double bond refers to one of thefollowing structures:

wherein R^(c) and R^(d) are, at each occurrence, independently H or asubstituent. For example, in some embodiments R^(c) and R^(d) are, ateach occurrence, independently H, C₁-C₁₂ alkyl or cycloalkyl, forexample H or C₁-C₁₂ alkyl.

In various other embodiments, the compound has one of the followingstructures (VIC) or (VID):

wherein e, f, g and h are each independently an integer from 1 to 12.

In some embodiments, the compound has structure (VIC). In otherembodiments, the compound has structure (VID).

In various embodiments of the compounds of structures (VIC) or (VID), e,f, g and h are each independently an integer from 4 to 10.

In other different embodiments,

or both, independently has one of the following structures:

In certain embodiments of the foregoing, a, b, c and d are eachindependently an integer from 2 to 12 or an integer from 4 to 12. Inother embodiments, a, b, c and d are each independently an integer from8 to 12 or 5 to 9. In some certain embodiments, a is 0. In someembodiments, a is 1. In other embodiments, a is 2. In more embodiments,a is 3. In yet other embodiments, a is 4. In some embodiments, a is 5.In other embodiments, a is 6. In more embodiments, a is 7. In yet otherembodiments, a is 8. In some embodiments, a is 9. In other embodiments,a is 10. In more embodiments, a is 11. In yet other embodiments, a is12. In some embodiments, a is 13. In other embodiments, a is 14. In moreembodiments, a is 15. In yet other embodiments, a is 16.

In some embodiments of structure (VI), b is 1. In other embodiments, bis 2. In more embodiments, b is 3. In yet other embodiments, b is 4. Insome embodiments, b is 5. In other embodiments, b is 6. In moreembodiments, b is 7. In yet other embodiments, b is 8. In someembodiments, b is 9. In other embodiments, b is 10. In more embodiments,b is 11. In yet other embodiments, b is 12. In some embodiments, b is13. In other embodiments, b is 14. In more embodiments, b is 15. In yetother embodiments, b is 16.

In some embodiments of structure (VI), c is 1. In other embodiments, cis 2. In more embodiments, c is 3. In yet other embodiments, c is 4. Insome embodiments, c is 5. In other embodiments, c is 6. In moreembodiments, c is 7. In yet other embodiments, c is 8. In someembodiments, c is 9. In other embodiments, c is 10. In more embodiments,c is 11. In yet other embodiments, c is 12. In some embodiments, c is13. In other embodiments, c is 14. In more embodiments, c is 15. In yetother embodiments, c is 16.

In some certain embodiments of structure (VI), d is 0. In someembodiments, d is 1. In other embodiments, d is 2. In more embodiments,d is 3. In yet other embodiments, d is 4. In some embodiments, d is 5.In other embodiments, d is 6. In more embodiments, d is 7. In yet otherembodiments, d is 8. In some embodiments, d is 9. In other embodiments,d is 10. In more embodiments, d is 11. In yet other embodiments, d is12. In some embodiments, d is 13. In other embodiments, d is 14. In moreembodiments, d is 15. In yet other embodiments, d is 16.

In some embodiments of structure (VI), e is 1. In other embodiments, eis 2. In more embodiments, e is 3. In yet other embodiments, e is 4. Insome embodiments, e is 5. In other embodiments, e is 6. In moreembodiments, e is 7. In yet other embodiments, e is 8. In someembodiments, e is 9. In other embodiments, e is 10. In more embodiments,e is 11. In yet other embodiments, e is 12.

In some embodiments of structure (VI), f is 1. In other embodiments, fis 2. In more embodiments, f is 3. In yet other embodiments, f is 4. Insome embodiments, f is 5. In other embodiments, f is 6. In moreembodiments, f is 7. In yet other embodiments, f is 8. In someembodiments, f is 9. In other embodiments, f is 10. In more embodiments,f is 11. In yet other embodiments, f is 12.

In some embodiments of structure (VI), g is 1. In other embodiments, gis 2. In more embodiments, g is 3. In yet other embodiments, g is 4. Insome embodiments, g is 5. In other embodiments, g is 6. In moreembodiments, g is 7. In yet other embodiments, g is 8. In someembodiments, g is 9. In other embodiments, g is 10. In more embodiments,g is 11. In yet other embodiments, g is 12.

In some embodiments of structure (VI), h is 1. In other embodiments, eis 2. In more embodiments, h is 3. In yet other embodiments, h is 4. Insome embodiments, e is 5. In other embodiments, h is 6. In moreembodiments, h is 7. In yet other embodiments, h is 8. In someembodiments, h is 9. In other embodiments, h is 10. In more embodiments,his 11. In yet other embodiments, his 12.

In some other various embodiments of structure (VI), a and d are thesame. In some other embodiments, b and c are the same. In some otherspecific embodiments a and d are the same and b and c are the same.

The sum of a and b and the sum of c and d are factors which may bevaried to obtain a lipid having the desired properties. In oneembodiment, a and b are chosen such that their sum is an integer rangingfrom 14 to 24. In other embodiments, c and d are chosen such that theirsum is an integer ranging from 14 to 24. In further embodiment, the sumof a and b and the sum of c and d are the same. For example, in someembodiments the sum of a and b and the sum of c and d are both the sameinteger which may range from 14 to 24. In still more embodiments, a. b,c and d are selected such that the sum of a and b and the sum of c and dis 12 or greater.

The substituents at R^(1a), R^(2a), R^(3a) and R^(4a) are notparticularly limited. In some embodiments, at least one of R^(1a),R^(2a), R^(3a) and R^(4a) is H. In certain embodiments R^(1a), R^(2a),R^(3a) and R^(4a) are H at each occurrence. In certain other embodimentsat least one of R^(1a), R^(2a), R^(3a) and R^(4a) is C₁-C₁₂ alkyl. Incertain other embodiments at least one of R^(1a), R^(2a), R^(3a) andR^(4a) is C₁-C₈ alkyl. In certain other embodiments at least one ofR^(1a), R^(2a), R^(3a) and R^(4a) is C₁-C₆ alkyl. In some of theforegoing embodiments, the C₁-C₈ alkyl is methyl, ethyl, n-propyl,iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

In certain embodiments of the foregoing, R^(1a), R^(1b), R^(4a) andR^(4b) are C₁-C₁₂ alkyl at each occurrence.

In further embodiments of the foregoing, at least one of R^(1b), R^(2b),R^(3b) and R^(4b) is H or R^(1b), R^(2b), R^(3b) and R^(4b) are H ateach occurrence.

In certain embodiments of the foregoing, R^(1b) together with the carbonatom to which it is bound is taken together with an adjacent R^(1b) andthe carbon atom to which it is bound to form a carbon-carbon doublebond. In other embodiments of the foregoing R^(4b) together with thecarbon atom to which it is bound is taken together with an adjacentR^(4b) and the carbon atom to which it is bound to form a carbon-carbondouble bond.

The substituents at R⁵ and R⁶ are not particularly limited in theforegoing embodiments. In certain embodiments one of R⁵ or R⁶ is methyl.In other embodiments each of R⁵ or R⁶ is methyl.

The substituents at R⁷ are not particularly limited in the foregoingembodiments. In certain embodiments R⁷ is C₆-C₁₆ alkyl. In some otherembodiments, R⁷ is C₆-C₉ alkyl. In some of these embodiments, R⁷ issubstituted with —(C═O)OR^(b), —O(C═O)R^(b), —C(═O)R^(b), —OR^(b),—S(O)_(x)R^(b), —S—SR^(b), —C(═O)SR^(b), —SC(═O)R^(b), —NR^(a)R^(b),—NR^(a)C(═O)R^(b), —C(═O)NR^(a)R^(b), —NR^(a)C(═O)NR^(a)R^(b),—OC(═O)NR^(a)R^(b), —NR^(a)C(═O)OR^(b), —NR^(a)S(O)_(x)NR^(a)R^(b),—NR^(a)S(O)_(x)R^(b) or —S(O)_(x)NR^(a)R^(b), wherein: R^(a) is H orC₁-C₁₂ alkyl; R^(b) is C₁-C₁₅ alkyl; and x is 0, 1 or 2. For example, insome embodiments R⁷ is substituted with —(C═O)OR^(b) or —O(C═O)R^(b).

In various of the foregoing embodiments of structure (VI), R^(b) isbranched C₃-C₁₅ alkyl. For example, in some embodiments R^(b) has one ofthe following structures:

In certain embodiments, R⁸ is OH.

In other embodiments of structure (VI), R⁸ is —N(R⁹)(C═O)R¹⁰. In someother embodiments, R⁸ is —(C═O)NR⁹R¹⁰. In still more embodiments, R⁸ is—NR⁹R¹⁰. In some of the foregoing embodiments, R⁹ and R¹⁰ are eachindependently H or C₁-C₈ alkyl, for example H or C₁-C₃ alkyl. In morespecific of these embodiments, the C₁-C₈ alkyl or C₁-C₃ alkyl isunsubstituted or substituted with hydroxyl. In other of theseembodiments, R⁹ and R¹⁰ are each methyl.

In yet more embodiments of structure (VI), R⁸ is —(C═O)OR¹¹. In some ofthese embodiments is benzyl.

In yet more specific embodiments of structure (VI), R⁸ has one of thefollowing structures:

In still other embodiments of the foregoing compounds, G³ is C₂-C₅alkylene, for example C₂-C₄ alkylene, C₃ alkylene or C₄ alkylene. Insome of these embodiments, R₈ is OH. In other embodiments, G² is absentand R⁷ is C₁-C₂ alkylene, such as methyl.

In various different embodiments, the compound has one of the structuresset forth in Table 5 below.

TABLE 5 Representative cationic lipids of structure (VI) No. StructureVI-1

VI-2

VI-3

VI-4

VI-5

VI-6

VI-7

VI-8

VI-9

VI-10

VI-11

VI-12

VI-13

VI-14

VI-15

VI-16

VI-17

VI-18

VI-19

VI-20

VI-21

VI-22

VI-23

VI-24

VI-25

VI-26

VI-27

VI-28

VI-29

VI-30

VI-31

VI-32

VI-33

VI-34

VI-35

VI-36

VI-37

In one embodiment, the cationic lipid is a compound having the followingstructure (VII):

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof,wherein:

-   -   X and X′ are each independently N or CR;    -   Y and Y′ are each independently absent, —O(C═O)—, —(C═O)O— or        NR, provided that:        -   a) Y is absent when X is N;        -   b) Y′ is absent when X′ is N;        -   c) Y is —O(C═O)—, —(C═O)O— or NR when X is CR; and        -   d) Y′ is —O(C═O)—, —(C═O)O— or NR when X′ is CR,    -   L¹ and L^(1′) are each independently —O(C═O)R¹, —(C═O)OR¹,        —C(═O)R¹, —OR¹, —S(O)_(z)R¹, —S—SR′, —C(═O)SR¹, —SC(═O)R¹,        —NR^(a)C(═O)R¹, —C(═O)NR^(b)R^(c), —NR^(a)C(═O)NR^(b)R^(c),        —OC(═O)NR^(b)R^(c) or —NR^(a)C(═O)OR¹;    -   L² and L^(2′) are each independently —O(C═O)R², —(C═O)OR²,        —C(═O)R², —OR², —S(O)_(z)R², —S—SR², —C(═O)SR², —SC(═O)R²,        —NR^(d)C(═O)R², —C(═O)NR^(c)R^(f), —NR^(d)C(═O)NR^(c)R^(f),        —OC(═O)NR^(c)R^(f), —NR^(d)C(═O)OR² or a direct bond to R²;    -   G¹, G^(1′), G² and G^(2′) are each independently C₂-C₁₂ alkylene        or C₂-C₁₂ alkenylene;    -   G³ is C₂-C₂₄ heteroalkylene or C₂-C₂₄ heteroalkenylene;    -   R^(a), R^(b), R^(d) and R^(e) are, at each occurrence,        independently H, C₁-C₁₂ alkyl or C₂-C₁₂ alkenyl;    -   R^(c) and R^(f) are, at each occurrence, independently C₁-C₁₂        alkyl or C₂-C₁₂ alkenyl;    -   R is, at each occurrence, independently H or C₁-C₁₂ alkyl;    -   R¹ and R² are, at each occurrence, independently branched C₆-C₂₄        alkyl or branched C₆-C₂₄ alkenyl;    -   z is 0, 1 or 2, and        wherein each alkyl, alkenyl, alkylene, alkenylene,        heteroalkylene and heteroalkenylene is independently substituted        or unsubstituted unless otherwise specified.

In other different embodiments of structure (VII):

-   -   X and X′ are each independently N or CR;    -   Y and Y′ are each independently absent or NR, provided that:        -   a) Y is absent when X is N;        -   b) Y′ is absent when X′ is N;        -   c) Y is NR when X is CR; and        -   d) Y′ is NR when X′ is CR,    -   L¹ and L′ are each independently —O(C═O)R¹, —(C═O)OR¹, —C(═O)R¹,        —OR¹, —S(O)_(z)R¹, —S—SR¹, —C(═O)SR¹, —SC(═O)R¹, —NR^(a)C(═O)R¹,        —C(═O)NR^(b)R^(c), —NR^(a)C(═O)NR^(b)R^(c), —OC(═O)NR^(b)R^(c)        or —NR^(a)C(═O)OR¹;    -   L² and L^(2′) are each independently —O(C═O)R², —(C═O)OR²,        —C(═O)R², —OR², —S(O)^(z)R², —S—SR², —C(═O)SR², —SC(═O)R²,        —NR^(d)C(═O)R², —C(═O)NR^(e)R^(f), —NR^(d)C(═O)NR^(e)R^(f),        —OC(═O)NR^(e)R^(f); —NR^(d)C(═O)OR² or a direct bond to R²;    -   G¹, G^(1′), G² and G^(2′) are each independently C₂-C₁₂ alkylene        or C₂-C₁₂ alkenylene;    -   G³ is C₂-C₂₄ alkyleneoxide or C₂-C₂₄ alkenyleneoxide;    -   R^(a), R^(b), R^(d) and R^(e) are, at each occurrence,        independently H, C₁-C₁₂ alkyl or C₂-C₁₂ alkenyl;    -   R^(c) and R^(f) are, at each occurrence, independently C₁-C₁₂        alkyl or C₂-C₁₂ alkenyl;    -   R is, at each occurrence, independently H or C₁-C₁₂ alkyl;    -   R¹ and R² are, at each occurrence, independently branched C₆-C₂₄        alkyl or branched C₆-C₂₄ alkenyl;    -   z is 0, 1 or 2, and        wherein each alkyl, alkenyl, alkylene, alkenylene, alkyleneoxide        and alkenyleneoxide is independently substituted or        unsubstituted unless otherwise specified.

In some embodiments of structure (VII), G³ is C₂-C₂₄ alkyleneoxide orC₂-C₂₄ alkenyleneoxide. In certain embodiments, G³ is unsubstituted. Inother embodiments, G³ is substituted, for example substituted withhydroxyl. In more specific embodiments G³ is C₂-C₁₂ alkyleneoxide, forexample, in some embodiments G³ is C₃-C₇ alkyleneoxide or in otherembodiments G³ is C₃-C₁₂ alkyleneoxide.

In other embodiments of structure (VII), G³ is C₂-C₂₄ alkyleneaminyl orC₂-C₂₄ alkenyleneaminyl, for example C₆-C₁₂ alkyleneaminyl. In some ofthese embodiments, G³ is unsubstituted. In other of these embodiments,G³ is substituted with C₁-C₆ alkyl.

In some embodiments of structure (VII), X and X′ are each N, and Y andY′ are each absent. In other embodiments, X and X′ are each CR, and Yand Y′ are each NR. In some of these embodiments, R is H.

In certain embodiments of structure (VII), X and X′ are each CR, and Yand Y′ are each independently —O(C═O)— or —(C═O)O—.

In some of the foregoing embodiments of structure (VII), the compoundhas one of the following structures (VIIA), (VIM), (VIIC), (VIID),(VIIF), (VIIG) or (VIIH):

wherein R^(d) is, at each occurrence, independently H or optionallysubstituted C₁-C₆ alkyl. For example, in some embodiments R^(d) is H. Inother embodiments, R^(d) is C₁-C₆ alkyl, such as methyl. In otherembodiments, R^(d) is substituted C₁-C₆ alkyl, such as C₁-C₆ alkylsubstituted with —O(C═O)R, —(C═O)OR, —NRC(═O)R or —C(═O)N(R)₂, wherein Ris, at each occurrence, independently H or C₁-C₁₂ alkyl.

In some of the foregoing embodiments of structure (VII), L¹ and L^(1′)are each independently —O(C═O)R¹, —(C═O)OR¹ or —C(═O)NR^(b)R^(c), and L²and L^(2′) are each independently —O(C═O)R², —(C═O)OR² or—C(═O)NR^(e)R^(f). For example, in some embodiments L¹ and L^(1′) areeach —(C═O)OR¹, and L² and L^(2′) are each —(C═O)OR². In otherembodiments L¹ and L^(1′) are each —(C═O)OR¹, and L² and L^(2′) are each—C(═O)NR^(e)R^(f). In other embodiments L¹ and L^(1′) are each—C(═O)NR^(b)R^(c), and L² and L^(2′) are each —C(═O)NR^(e)R^(f).

In some embodiments of the foregoing, G¹, G^(1′), G² and G^(2′) are eachindependently C₂-C₈ alkylene, for example C₄-C₈ alkylene.

In some of the foregoing embodiments of structure (VII), R¹ or R², areeach, at each occurrence, independently branched C₆-C₂₄ alkyl. Forexample, in some embodiments, R¹ and R² at each occurrence,independently have the following structure:

wherein:

-   -   R^(7a) and R^(7b) are, at each occurrence, independently H or        C₁-C₁₂ alkyl; and    -   a is an integer from 2 to 12,        wherein R^(7a), R^(7b) and a are each selected such that R¹ and        R² each independently comprise from 6 to 20 carbon atoms. For        example, in some embodiments a is an integer ranging from 5 to 9        or from 8 to 12.

In some of the foregoing embodiments of structure (VII), at least oneoccurrence of R^(7a) is H. For example, in some embodiments, R^(7a) is Hat each occurrence. In other different embodiments of the foregoing, atleast one occurrence of R^(7b) is C₁-C₈ alkyl. For example, in someembodiments, C₁-C₈ alkyl is methyl, ethyl, n-propyl, iso-propyl,n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

In different embodiments of structure (VII), R¹ or R², or both, at eachoccurrence independently has one of the following structures:

In some of the foregoing embodiments of structure (VII), R^(b), R^(c),R^(e) and R^(f), when present, are each independently C₃-C₁₂ alkyl. Forexample, in some embodiments R^(b), R^(c), R^(e) and R^(f), whenpresent, are n-hexyl and in other embodiments R^(b), R^(c), R^(e) andR^(f), when present, are n-octyl.

In various different embodiments of structure (VII), the cationic lipidhas one of the structures set forth in Table 6 below.

TABLE 6 Representative cationic lipids of structure (VII) No. StructureVII-1

VII-2

VII-3

VII-4

VII-5

VII-6

VII-7

VII-8

VII-9

VII-10

VII-11

In one embodiment, the cationic lipid is a compound having the followingstructure (VIII):

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof,wherein:

-   -   X is N, and Y is absent; or X is CR, and Y is NR;    -   L¹ is —O(C═O)R¹, —(C═O)OR¹, —C(═O)R¹, —OR¹, —S(O)_(x)R¹, —S—SR¹,        —C(═O)SR¹, —SC(═O)R¹, —NR^(a)C(═O)R¹, —C(═O)NR^(b)R^(c),        —NR^(a)C(═O)NR^(b)R^(c), —OC(═O)NR^(b)R^(c) or —NR^(a)C(═O)OR¹;    -   L² is —O(C═O)R², —(C═O)OR², —C(═O)R², —OR², —S(O)_(x)R², —S—SR²,        —C(═O)SR², —SC(═O)R², —NR^(d)C(═O)R², —C(═O)NR^(e)R^(f),        —NR^(d)C(═O)NR^(e)R^(f), —OC(═O)NR^(e)R^(f); —NR^(d)C(═O)OR² or        a direct bond to R²;    -   L³ is —O(C═O)R³ or —(C═O)OR³;    -   G¹ and G² are each independently C₂-C₁₂ alkylene or C₂-C₁₂        alkenylene;    -   G³ is C₁-C₂₄ alkylene, C₂-C₂₄ alkenylene, C₁-C₂₄ heteroalkylene        or C₂-C₂₄ heteroalkenylene;    -   R^(a), R^(b), R^(d) and R^(e) are each independently H or C₁-C₁₂        alkyl or C₁-C₁₂ alkenyl;    -   R^(c) and R^(f) are each independently C₁-C₁₂ alkyl or C₂-C₁₂        alkenyl;    -   each R is independently H or C₁-C₁₂ alkyl;    -   R¹, R² and R³ are each independently C₁-C₂₄ alkyl or C₂-C₂₄        alkenyl; and    -   x is 0, 1 or 2, and    -   wherein each alkyl, alkenyl, alkylene, alkenylene,        heteroalkylene and heteroalkenylene is independently substituted        or unsubstituted unless otherwise specified.

In more embodiments of structure (I):

-   -   X is N, and Y is absent; or X is CR, and Y is NR;    -   L¹ is —O(C═O)R¹, —(C═O)OR¹, —C(═O)R¹, —OR¹, —S(O)_(x)R¹, —S—SR¹,        —C(═O)SR¹, —SC(═O)R¹, —NR^(a)C(═O)R¹, —C(═O)NR^(b)R^(c),        —NR^(a)C(═O)NR^(b)R^(c), —OC(═O)NR^(b)R^(c) or —NR^(a)C(═O)OR¹;    -   L² is —O(C═O)R₂, —(C═O)OR², —C(═O)R², —OR², —S(O)_(x)R², —S—SR²,        —C(═O)SR², —SC(═O)R², —NR^(d)C(═O)R², —C(═O)NR^(e)R^(f),        —NR^(d)C(═O)NR^(e)R^(f), —OC(═O)NR^(c)R^(f); —NR^(d)C(═O)OR² or        a direct bond to R²;    -   L³ is —O(C═O)R³ or —(C═O)OR³;    -   G¹ and G² are each independently C₂-C₁₂ alkylene or C₂-C₁₂        alkenylene;    -   G³ is C₁-C₂₄ alkylene, C₂-C₂₄ alkenylene, C₁-C₂₄ heteroalkylene        or C₂-C₂₄ heteroalkenylene when X is CR, and Y is NR; and G³ is        C₁-C₂₄ heteroalkylene or C₂-C₂₄ heteroalkenylene when X is N,        and Y is absent;    -   R^(a), R^(b), R^(d) and R^(e) are each independently H or C₁-C₁₂        alkyl or C₁-C₁₂ alkenyl;    -   R^(c) and R^(f) are each independently C₁-C₁₂ alkyl or C₂-C₁₂        alkenyl;    -   each R is independently H or C₁-C₁₂ alkyl;    -   R¹, R² and R³ are each independently C₁-C₂₄ alkyl or C₂-C₂₄        alkenyl; and    -   x is 0, 1 or 2, and    -   wherein each alkyl, alkenyl, alkylene, alkenylene,        heteroalkylene and heteroalkenylene is independently substituted        or unsubstituted unless otherwise specified.

In other embodiments of structure (I):

-   -   X is N and Y is absent, or X is CR and Y is NR;    -   L¹ is —O(C═O)R¹, —(C═O)OR¹, —C(═O)R¹, —OR¹, —S(O)_(x)R¹, —S—SR¹,        —C(═O)SR¹, —SC(═O)R¹, —NR^(a)C(═O)R¹, —C(═O)NR^(b)R^(c),        —NR^(a)C(═O)NR^(b)R^(c), —OC(═O)NR^(b)R^(c) or —NR^(a)C(═O)OR¹;    -   L² is —O(C═O)R², —(C═O)OR², —C(═O)R², —OR², —S(O)_(x)R², —S—SR²,        —C(═O)SR², —SC(═O)R², —NR^(d)C(═O)R², —C(═O)NR^(e)R^(f),        —NR^(d)C(═O)NR^(e)R^(f), —OC(═O)NR^(e)R^(f); —NR^(d)C(═O)OR² or        a direct bond to R²;    -   L³ is —O(C═O)R³ or —(C═O)OR³;    -   G¹ and G² are each independently C₂-C₁₂ alkylene or C₂-C₁₂        alkenylene;    -   G³ is C₁-C₂₄ alkylene, C₂-C₂₄ alkenylene, C₁-C₂₄ heteroalkylene        or C₂-C₂₄ heteroalkenylene;    -   R^(a), R^(b), R^(d) and R^(e) are each independently H or C₁-C₁₂        alkyl or C₁-C₁₂ alkenyl;    -   R^(c) and R^(f) are each independently C₁-C₁₂ alkyl or C₂-C₁₂        alkenyl;    -   each R is independently H or C₁-C₁₂ alkyl;    -   R¹, R² and R³ are each independently branched C₆-C₂₄ alkyl or        branched C₆-C₂₄ alkenyl; and    -   x is 0, 1 or 2, and        wherein each alkyl, alkenyl, alkylene, alkenylene,        heteroalkylene and heteroalkenylene is independently substituted        or unsubstituted unless otherwise specified.

In certain embodiments of structure (VIII), G³ is unsubstituted. In morespecific embodiments G³ is C₂-C₁₂ alkylene, for example, in someembodiments G³ is C₃-C₇ alkylene or in other embodiments G³ is C₃-C₁₂alkylene. In some embodiments, G³ is C₂ or C₃ alkylene.

In other embodiments of structure (VIII), G³ is C₁-C₁₂ heteroalkylene,for example C₁-C₁₂ aminylalkylene.

In certain embodiments of structure (VIII), X is N and Y is absent. Inother embodiments, X is CR and Y is NR, for example in some of theseembodiments R is H.

In some of the foregoing embodiments of structure (VIII), the compoundhas one of the following structures (VIIIA), (VIIIB), (VIIIC) or (VIIID:

In some of the foregoing embodiments of structure (VIII), L¹ is—O(C═O)R¹, —(C═O)OR¹ or —C(═O)NR^(b)R^(c), and L² is —O(C═O)R²,—(C═O)OR² or —C(═O)NR^(e)R^(f). In other specific embodiments, L¹ is—(C═O)OR¹ and L² is —(C═O)OR². In any of the foregoing embodiments, L³is —(C═O)OR³.

In some of the foregoing embodiments of structure (VIII), G¹ and G² areeach independently C₂-C₁₂ alkylene, for example C₄-C₁₀ alkylene.

In some of the foregoing embodiments of structure (VIII), R¹, R² and R³are each, independently branched C₆-C₂₄ alkyl. For example, in someembodiments, R¹, R² and R³ each, independently have the followingstructure:

wherein:

-   -   R^(7a) and R^(7b) are, at each occurrence, independently H or        C₁-C₁₂ alkyl; and    -   a is an integer from 2 to 12,        wherein R^(7a), R^(7b) and a are each selected such that R¹ and        R² each independently comprise from 6 to 20 carbon atoms. For        example, in some embodiments a is an integer ranging from 5 to 9        or from 8 to 12.

In some of the foregoing embodiments of structure (VIII), at least oneoccurrence of R^(7a) is H. For example, in some embodiments, R^(7a) is Hat each occurrence.

In other different embodiments of the foregoing, at least one occurrenceof R^(7b) is C₁-C₈ alkyl. For example, in some embodiments, C₁-C₈ alkylis methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl,n-hexyl or n-octyl.

In some of the foregoing embodiments of structure (VIII), X is CR, Y isNR and R³ is C₁-C₁₂ alkyl, such as ethyl, propyl or butyl. In some ofthese embodiments, R¹ and R² are each independently branched C₆-C₂₄alkyl.

In different embodiments of structure (VIII), R¹, R² and R³ each,independently have one of the following structures:

In certain embodiments of structure (VIII), R¹ and R² and R³ are each,independently, branched C₆-C₂₄ alkyl and R³ is C₁-C₂₄ alkyl or C₂-C₂₄alkenyl.

In some of the foregoing embodiments of structure (VIII), R^(b), R^(c),R^(e) and R^(f) are each independently C₃-C₁₂ alkyl. For example, insome embodiments R^(b), R^(c), R^(e) and R^(f) are n-hexyl and in otherembodiments R^(b), R^(c), R^(e) and R^(f) are n-octyl.

In various different embodiments of structure (VIII), the compound hasone of the structures set forth in Table 7 below.

TABLE 7 Representative cationic lipids of structure (VIII) No. StructureVIII-1

VIII-2

VIII-3

VIII-4

VIII-5

VIII-6

VIII-7

VIII-8

VIII-9

VIII-10

VIII-11

VIII-12

In one embodiment, the cationic lipid is a compound having the followingstructure (IX):

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof,wherein:

-   -   L¹ is —O(C═O)R¹, —(C═O)OR¹, —C(═O)R¹, —OR¹, —S(O)_(x)R¹, —S—SR¹,        —C(═O)SR¹, —SC(═O)R¹, —NR^(a)C(═O)R¹, —C(═O)NR^(b),        —NR^(a)C(═O)NR^(b)R^(c), —OC(═O)NR^(b)R^(c) or —NR^(a)C(═O)OR¹;    -   L² is —O(C═O)R², —(C═O)OR², —C(═O)R², —OR², —S(O), R², —S—SR²,        —C(═O)SR², —SC(═O)R², —NR^(d)C(═O)R², —C(═O)NR^(e)R^(f),        —NR^(d)C(═O)NR^(e)R^(f), —OC(═O)NR^(e)R^(f), —NR^(d)C(═O)OR² or        a direct bond to R²;    -   G¹ and G² are each independently C₂-C₁₂ alkylene or C₂-C₁₂        alkenylene;    -   G³ is C₁-C₂₄ alkylene, C₂-C₂₄ alkenylene, C₃-C₅ cycloalkylene or        C₃-C₅ cycloalkenylene,    -   R^(a), R^(b), R^(d) and R^(e) are each independently H or C₁-C₁₂        alkyl or C₁-C₁₂ alkenyl;    -   R^(c) and R^(f) are each independently C₁-C₁₂ alkyl or C₂-C₁₂        alkenyl;    -   R¹ and R² are each independently branched C₆-C₂₄ alkyl or        branched C₆-C₂₄ alkenyl;    -   R³ is —N(R⁴)R⁵;    -   R⁴ is C₁-C₁₂ alkyl;    -   R⁵ is substituted C₁-C₁₂ alkyl; and    -   x is 0, 1 or 2, and        wherein each alkyl, alkenyl, alkylene, alkenylene,        cycloalkylene, cycloalkenylene, aryl and aralkyl is        independently substituted or unsubstituted unless otherwise        specified.

In certain embodiments of structure (XI), G³ is unsubstituted. In morespecific embodiments G³ is C₂-C₁₂ alkylene, for example, in someembodiments G³ is C₃-C₇ alkylene or in other embodiments G³ is C₃-C₁₂alkylene. In some embodiments, G³ is C₂ or C₃ alkylene.

In some of the foregoing embodiments of structure (IX), the compound hasthe following structure (IX A):

wherein y and z are each independently integers ranging from 2 to 12,for example an integer from 2 to 6, from 4 to 10, or for example 4 or 5.In certain embodiments, y and z are each the same and selected from 4,5, 6, 7, 8 and 9.

In some of the foregoing embodiments of structure (IX), L¹ is —O(C═O)R¹,—(C═O)OR¹ or —C(═O)NR^(b)R^(c), and L² is —O(C═O)R², —(C═O)OR² or—C(═O)NR^(e)R^(f). For example, in some embodiments L¹ and L² are—(C═O)OR¹ and —(C═O)OR², respectively. In other embodiments L¹ is—(C═O)OR¹ and L² is —C(═O)NR^(e)R^(f). In other embodiments L¹ is—C(═O)NR^(b)R^(c) and L² is —C(═O)NR^(e)R^(f).

In other embodiments of the foregoing, the compound has one of thefollowing structures (IXB), (IXC), (IXD) or (IXE):

In some of the foregoing embodiments, the compound has structure (IXB),in other embodiments, the compound has structure (IXC) and in stillother embodiments the compound has the structure (IXD). In otherembodiments, the compound has structure (IXE).

In some different embodiments of the foregoing, the compound has one ofthe following structures (IXF), (IXG), (IXH) or (IXJ):

wherein y and z are each independently integers ranging from 2 to 12,for example an integer from 2 to 6, for example 4.

In some of the foregoing embodiments of structure (IX), y and z are eachindependently an integer ranging from 2 to 10, 2 to 8, from 4 to 10 orfrom 4 to 7. For example, in some embodiments, y is 4, 5, 6, 7, 8, 9,10, 11 or 12. In some embodiments, z is 4, 5, 6, 7, 8, 9, 10, 11 or 12.In some embodiments, y and z are the same, while in other embodiments yand z are different.

In some of the foregoing embodiments of structure (IX), R¹ or R², orboth is branched C₆-C₂₄ alkyl. For example, in some embodiments, R¹ andR² each, independently have the following structure:

wherein:

-   -   R^(7a) and R^(7b) are, at each occurrence, independently H or        C₁-C₁₂ alkyl; and    -   a is an integer from 2 to 12,        wherein R^(7a), R^(7b) and a are each selected such that R¹ and        R² each independently comprise from 6 to 20 carbon atoms. For        example, in some embodiments a is an integer ranging from 5 to 9        or from 8 to 12.

In some of the foregoing embodiments of structure (IX), at least oneoccurrence of R^(7a) is H. For example, in some embodiments, R^(7a) is Hat each occurrence. In other different embodiments of the foregoing, atleast one occurrence of R^(7b) is C₁-C₈ alkyl. For example, in someembodiments, C₁-C₈ alkyl is methyl, ethyl, n-propyl, iso-propyl,n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

In different embodiments of structure (IX), R¹ or R², or both, has oneof the following structures:

In some of the foregoing embodiments of structure (IX), R^(b), R^(c),R^(e) and R^(f) are each independently C₃-C₁₂ alkyl. For example, insome embodiments R^(b), R^(c), R^(e) and R^(f) are n-hexyl and in otherembodiments R^(b), R^(c), R^(e) and R^(f) are n-octyl.

In any of the foregoing embodiments of structure (IX), R⁴ is substitutedor unsubstituted: methyl, ethyl, propyl, n-butyl, n-hexyl, n-octyl orn-nonyl. For example, in some embodiments R⁴ is unsubstituted. In otherR⁴ is substituted with one or more substituents selected from the groupconsisting of —OR^(g), —NR^(g)C(═O)R^(h), —C(═O)NR^(g)R^(h),—C(═O)R^(h), —OC(═O)R^(h), —C(═O)OR^(h) and —OR^(i)OH, wherein:

-   -   R^(g) is, at each occurrence independently H or C₁-C₆ alkyl;    -   R^(h) is at each occurrence independently C₁-C₆ alkyl; and    -   R^(i) is, at each occurrence independently C₁-C₆ alkylene.

In other of the foregoing embodiments of structure (IX), R⁵ issubstituted: methyl, ethyl, propyl, n-butyl, n-hexyl, n-octyl orn-nonyl. In some embodiments, R⁵ is substituted ethyl or substitutedpropyl. In other different embodiments, R⁵ is substituted with hydroxyl.In still more embodiments, R⁵ is substituted with one or moresubstituents selected from the group consisting of —OR^(g),—NR^(g)C(═O)R^(h), —C(═O)NR^(g)R^(h), —C(═O)R^(h), —OC(═O)R^(h),—C(═O)OR^(h) and —OR^(i)OH, wherein:

-   -   R^(g) is, at each occurrence independently H or C₁-C₆ alkyl;    -   R^(h) is at each occurrence independently C₁-C₆ alkyl; and    -   R^(i) is, at each occurrence independently C₁-C₆ alkylene.

In other embodiments of structure (IX), R⁴ is unsubstituted methyl, andR⁵ is substituted: methyl, ethyl, propyl, n-butyl, n-hexyl, n-octyl orn-nonyl. In some of these embodiments, R⁵ is substituted with hydroxyl.

In some other specific embodiments of structure (IX), R³ has one of thefollowing structures:

In various different embodiments of structure (IX), the cationic lipidhas one of the structures set forth in Table 8 below.

TABLE 8 Representative cationic lipids of structure (IX) No. StructureIX-1

IX-2

IX-3

IX-4

IX-5

IX-6

IX-7

IX-8

IX-9

IX-10

IX-11

IX-12

IX-13

IX-14

IX-15

IX-16

IX-17

IX-18

In one embodiment, the cationic lipid is a compound having the followingstructure (X):

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomerthereof, wherein:

-   -   G¹ is —OH, —NR³R⁴, —(C═O)NR⁵ or —NR³(C═O)R⁵;    -   G² is —CH₂— or —(C═O)—;    -   R is, at each occurrence, independently H or OH;    -   R¹ and R² are each independently branched, saturated or        unsaturated C₁₂-C₃₆ alkyl;    -   R³ and R⁴ are each independently H or straight or branched,        saturated or unsaturated C₁-C₆ alkyl;    -   R⁵ is straight or branched, saturated or unsaturated C₁-C₆        alkyl; and    -   n is an integer from 2 to 6.

In some embodiments, R¹ and R² are each independently branched,saturated or unsaturated C₁₂-C₃₀ alkyl, C₁₂-C₂₀ alkyl, or C₁₅-C₂₀ alkyl.In some specific embodiments, R¹ and R² are each saturated. In certainembodiments, at least one of R¹ and R² is unsaturated.

In some of the foregoing embodiments of structure (X), R¹ and R² havethe following structure:

In some of the foregoing embodiments of structure (X), the compound hasthe following structure (XA):

wherein:

-   -   R⁶ and R⁷ are, at each occurrence, independently H or straight        or branched, saturated or unsaturated C₁-C₁₄ alkyl;    -   a and b are each independently an integer ranging from 1 to 15,    -   provided that R⁶ and a, and R⁷ and b, are each independently        selected such that R¹ and R², respectively, are each        independently branched, saturated or unsaturated C₁₂-C₃₆ alkyl.

In some of the foregoing embodiments, the compound has the followingstructure (XB)

wherein:

-   -   R⁸, R⁹, R¹⁰ and R¹¹ are each independently straight or branched,        saturated or unsaturated C₄-C₁₂ alkyl, provided that R⁸ and R⁹,        and R¹⁰ and R¹¹, are each independently selected such that R¹        and R², respectively, are each independently branched, saturated        or unsaturated C₁₂-C₃₆ alkyl. In some embodiments of (XB), R⁸,        R⁹, R¹⁰ and R¹¹ are each independently straight or branched,        saturated or unsaturated C₆-C₁₀ alkyl. In certain embodiments of        (XB), at least one of R⁸, R⁹, R¹⁹ and R¹¹ is unsaturated. In        other certain specific embodiments of (XB), each of R⁸, R⁹, R¹⁰        and R¹¹ is saturated.

In some of the foregoing embodiments, the compound has structure (XA),and in other embodiments, the compound has structure (XB).

In some of the foregoing embodiments, G¹ is —OH, and in some embodimentsG¹ is —NR³R⁴. For example, in some embodiments, G¹ is —NH₂, —NHCH₃ or—N(CH₃)₂. In certain embodiments, G¹ is —(C═O)NR⁵. In certain otherembodiments, G¹ is —NR³(C═O)R⁵. For example, in some embodiments G¹ is—NH(C═O)CH₃ or —NH(C═O)CH₂CH₂CH₃.

In some of the foregoing embodiments of structure (X), G² is —CH₂—. Insome different embodiments, G² is —(C═O)—.

In some of the foregoing embodiments of structure (X), n is an integerranging from 2 to 6, for example, in some embodiments n is 2, 3, 4, 5 or6. In some embodiments, n is 2. In some embodiments, n is 3. In someembodiments, n is 4.

In certain of the foregoing embodiments of structure (X), at least oneof R¹, R², R³, R⁴ and R⁵ is unsubstituted. For example, in someembodiments, R¹, R², R³, R⁴ and R⁵ are each unsubstituted. In someembodiments, R³ is substituted. In other embodiments R⁴ is substituted.In still more embodiments, R⁵ is substituted. In certain specificembodiments, each of R³ and R⁴ are substituted. In some embodiments, asubstituent on R³, R⁴ or R⁵ is hydroxyl. In certain embodiments, R³ andR⁴ are each substituted with hydroxyl.

In some of the foregoing embodiments of structure (X), at least one R isOH. In other embodiments, each R is H.

In various different embodiments of structure (X), the compound has oneof the structures set forth in Table 9 below.

TABLE 9 Representative cationic lipids of structure (X) No. StructureX-1

X-2

X-3

X-4

X-5

X-6

X-7

X-8

X-9

X-10

X-11

X-12

X-13

X-14

X-15

X-16

X-17

In any of Embodiments 1, 2, 3, 4 or 5, the LNPs further comprise aneutral lipid. In various embodiments, the molar ratio of the cationiclipid to the neutral lipid ranges from about 2:1 to about 8:1. Incertain embodiments, the neutral lipid is present in any of theforegoing LNPs in a concentration ranging from 5 to 10 mol percent, from5 to 15 mol percent, 7 to 13 mol percent, or 9 to 11 mol percent. Incertain specific embodiments, the neutral lipid is present in aconcentration of about 9.5, 10 or 10.5 mol percent. In some embodiments,the molar ratio of cationic lipid to the neutral lipid ranges from about4.1:1.0 to about 4.9:1.0, from about 4.5:1.0 to about 4.8:1.0, or fromabout 4.7:1.0 to 4.8:1.0. In some embodiments, the molar ratio of totalcationic lipid to the neutral lipid ranges from about 4.1:1.0 to about4.9:1.0, from about 4.5:1.0 to about 4.8:1.0, or from about 4.7:1.0 to4.8:1.0.

Exemplary neutral lipids for use in any of Embodiments 1, 2, 3, 4 or 5include, for example, distearoylphosphatidylcholine (DSPC),dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine(DPPC), dioleoylphosphatidylglycerol (DOPG),dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE) anddioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE, 1-stearioyl-2-oleoylphosphatidyethanolamine (SOPE), and 1,2-dielaidoyl-sn-glycero-3-phophoethanolamine(transDOPE). In one embodiment, the neutral lipid is1,2-distearoyl-sn-glycero-3phosphocholine (DSPC). In some embodiments,the neutral lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPEand SM. In some embodiments, the neutral lipid is DSPC.

In various embodiments of Embodiments 1, 2, 3, 4 or 5, any of thedisclosed lipid nanoparticles comprise a steroid or steroid analogue. Incertain embodiments, the steroid or steroid analogue is cholesterol. Insome embodiments, the steroid is present in a concentration ranging from39 to 49 molar percent, 40 to 46 molar percent, from 40 to 44 molarpercent, from 40 to 42 molar percent, from 42 to 44 molar percent, orfrom 44 to 46 molar percent. In certain specific embodiments, thesteroid is present in a concentration of 40, 41, 42, 43, 44, 45, or 46molar percent.

In certain embodiments, the molar ratio of cationic lipid to the steroidranges from 1.0:0.9 to 1.0:1.2, or from 1.0:1.0 to 1.0:1.2. In some ofthese embodiments, the molar ratio of cationic lipid to cholesterolranges from about 5:1 to 1:1. In certain embodiments, the steroid ispresent in a concentration ranging from 32 to 40 mol percent of thesteroid.

In certain embodiments, the molar ratio of total cationic to the steroidranges from 1.0:0.9 to 1.0:1.2, or from 1.0:1.0 to 1.0:1.2. In some ofthese embodiments, the molar ratio of total cationic lipid tocholesterol ranges from about 5:1 to 1:1. In certain embodiments, thesteroid is present in a concentration ranging from 32 to 40 mol percentof the steroid.

In some embodiments of Embodiments 1, 2, 3 4 or 5, the LNPs furthercomprise a polymer conjugated lipid. In various other embodiments ofEmbodiments 1, 2, 3 4 or 5, the polymer conjugated lipid is a pegylatedlipid. For example, some embodiments include a pegylated diacylglycerol(PEG-DAG) such as1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), apegylated phosphatidylethanoloamine (PEG-PE), a PEG succinatediacylglycerol (PEG-S-DAG) such as4-O-(2′,3′-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanedioate(PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEGdialkoxypropylcarbamate such asω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or2,3-di(tetradecanoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate.

In various embodiments, the polymer conjugated lipid is present in aconcentration ranging from 1.0 to 2.5 molar percent. In certain specificembodiments, the polymer conjugated lipid is present in a concentrationof about 1.7 molar percent. In some embodiments, the polymer conjugatedlipid is present in a concentration of about 1.5 molar percent.

In certain embodiments, the molar ratio of cationic lipid to the polymerconjugated lipid ranges from about 35:1 to about 25:1. In someembodiments, the molar ratio of cationic lipid to polymer conjugatedlipid ranges from about 100:1 to about 20:1.

In certain embodiments, the molar ratio of total cationic lipid (i.e.,the sum of the first and second cationic lipid) to the polymerconjugated lipid ranges from about 35:1 to about 25:1. In someembodiments, the molar ratio of total cationic lipid to polymerconjugated lipid ranges from about 100:1 to about 20:1.

In some embodiments of Embodiments 1, 2, 3 4 or 5, the pegylated lipid,when present, has the following Formula (XI):

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,wherein:

-   -   R¹² and R¹³ are each independently a straight or branched,        saturated or unsaturated alkyl chain containing from 10 to 30        carbon atoms, wherein the alkyl chain is optionally interrupted        by one or more ester bonds; and    -   w has a mean value ranging from 30 to 60.

In some embodiments, R¹² and R¹³ are each independently straight,saturated alkyl chains containing from 12 to 16 carbon atoms. In otherembodiments, the average w ranges from 42 to 55, for example, theaverage w is 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55.In some specific embodiments, the average w is about 49.

In some embodiments, the pegylated lipid has the following Formula(XIa):

wherein the average w is about 49.

In some embodiments of Embodiments 1, 2, 3 4 or 5, the nucleic acid isselected from antisense and messenger RNA. For example, messenger RNAmay be used to induce an immune response (e.g., as a vaccine), forexample by translation of immunogenic proteins.

In other embodiments of Embodiments 1, 2, 3 4 or 5, the nucleic acid ismRNA, and the mRNA to lipid ratio in the LNP N/P, were N represents themoles of cationic lipid and P represents the moles of phosphate presentas part of the nucleic

In an embodiment, the transfer vehicle comprises a lipid or an ionizablelipid described in US patent publication number 20190314524.

Some embodiments of the present invention provide nucleic acid-lipidnanoparticle compositions comprising one or more of the novel cationiclipids described herein as structures listed in Table 10, that provideincreased activity of the nucleic acid and improved tolerability of thecompositions in vivo.

In one embodiment, an ionizable lipid has the following structure (XII):

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomerthereof, wherein:

-   -   one of L¹ or L² is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)—,        —S—S—, —C(═O)S—, SC(═O)—, —NR^(a)C(═O)—, —C(═O)NR^(a)—,        NR^(a)C(═)NR^(a)—, —OC(═O)NR^(a)— or —NR^(a)C(═O)O—, and the        other of L¹ or L² is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—,        —S(O)_(x)—, —S—S—, —C(═O)S—, SC(═O)—, —NR^(a)C(═O)—,        —C(═O)NR^(a)—, NR^(a)C(═O)NR^(a)—, —OC(═O)NR^(a)— or        —NR^(a)C(═O)O— or a direct bond;    -   G¹ and G² are each independently unsubstituted C₁-C₁₂alkylene or        C₁-C₁₂alkenylene;    -   G³ is C₁-C₂₄ alkylene, C₁-C₂₄ alkenylene, C₃-C₈ cycloalkylene,        C₃-C₈ cycloalkenylene;    -   R^(a) is H or C₁-C₁₂alkyl;    -   R¹ and R² are each independently C₆-C₂₄ alkyl or C₆-C₂₄ alkenyl;    -   R₃ is H, OR⁵, CN, —C(═O)OR⁴, —OC(═O)R₄ or —NR⁵C(═O)R⁴;    -   R⁴ is C₁-C₁₂alkyl;    -   R⁵ is H or C₁-C₆ alkyl; and    -   x is 0, 1 or 2.

In some embodiments, an ionizable lipid has one of the followingstructures (XIIA) or (XIIB):

wherein:

-   -   A is a 3 to 8-membered cycloalkyl or cycloalkylene ring;    -   R⁶ is, at each occurrence, independently H, OH or C₁-C₂₄ alkyl;        and    -   n is an integer ranging from 1 to 15.

In some embodiments, the ionizable lipid has structure (XIIA), and inother embodiments, the ionizable lipid has structure (XIIB).

In other embodiments, an ionizable lipid has one of the followingstructures (XIIC) or (XIID): (XIIC)

wherein y and z are each independently integers ranging from 1 to 12.

In some embodiments, one of L¹ or L² is —O(C═O)—. For example, in someembodiments each of L¹ and L² are —O(C═O)—. In some differentembodiments of any of the foregoing, L¹ and U are each independently—(C══O)O— or —O(C═O)—. For example, in some embodiments each of L¹ and Lis —(C═O)O—.

In some embodiments, an ionizable lipid has one of the followingstructures (XIIE) or (XIIF):

In some embodiments, an ionizable lipid has one of the followingstructures (XIIG), (XIIH), (XIII), or (XIIJ):

In some embodiments, n is an integer ranging from 2 to 12, for examplefrom 2 to 8 or from 2 to 4. For example, in some embodiments, n is 3, 4,5 or 6. In some embodiments, n is 3. In some embodiments, n is 4. Insome embodiments, n is 5. In some embodiments, n is 6.

In some embodiments, y and z are each independently an integer rangingfrom 2 to 10. For example, in some embodiments, y and z are eachindependently an integer ranging from 4 to 9 or from 4 to 6.

In some embodiments, R⁶ is H. In other embodiments. R⁶ is C₁-C₂₄ alkyl.In other embodiments, R⁶ is OH.

In some embodiments, G³ is unsubstituted. In other embodiments, G³ issubstituted. In various different embodiments, G³ is linear C₁-C₂₄alkylene or linear C₁-C₂₄ alkenylene.

In some embodiments, R¹ or R², or both, is C₆-C₂₄ alkenyl. For example,in some embodiments, R¹ and R² each, independently have the followingstructure:

wherein:

-   -   R^(7a) and R^(7b) are, at each occurrence, independently H or        C₁-C₁₂ alkyl; and    -   a is an integer from 2 to 12,    -   wherein R^(7a), R^(7b) and a are each selected such that R¹ and        R² each independently comprise from 6 to 20 carbon atoms.

In some embodiments, a is an integer ranging from 5 to 9 or from 8 to12.

In some embodiments, at least one occurrence of R^(7a) is H. Forexample, in some embodiments, R^(7a) is H at each occurrence. In otherdifferent embodiments, at least one occurrence of R^(7b) is C₁-C₈ alkyl.For example, in some embodiments, C₁-C₈ alkyl is methyl, ethyl,n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl orn-octyl.

In different embodiments, R¹ or R², or both, has one of the followingstructures:

In some embodiments, R³ is —OH, —CN, —C(═O)OR⁴, —OC(═O)R⁴ or —NHC(═O)R⁴.In some embodiments, R⁴ is methyl or ethyl.

In some embodiments, an ionizable lipid is a compound of Formula (1):

wherein:

-   -   each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,        13, 14, or 15; and    -   L₁ and L₃ are each independently —OC(O)—* or —C(O)O—*, wherein        “*” indicates the attachment point to R¹ or R₃;    -   R₁ and R₃ are each independently a linear or branched C₉-C₂₀        alkyl or C₉-C₂₀ alkenyl, optionally substituted by one or more        substituents selected from oxo, halo, hydroxy, cyano, alkyl,        alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl,        dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl,        alkylaminoalkyl, dialkylaminoalkyl,        (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl,        alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino,        aminoalkylcarbonylamino, aminocarbonylalkylamino,        (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino,        hydroxycarbonyl, alkyloxycarbonyl, aminocarbonyl,        aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl,        dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl,        (alkylaminoalkyl) (alkyl)aminocarbonyl, alkylaminoalkylcarbonyl,        dialkylaminoalkylcarbonyl, heterocyclylcarbonyl,        alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide,        alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl.

In some embodiments, R₁ and R₃ are the same. In some embodiments, R₁ andR₃ are different.

In some embodiments, R₁ and R₃ are each independently a branchedsaturated C₉-C₂₀ alkyl. In some embodiments, one of R₁ and R₃ is abranched saturated C₉-C₂₀ alkyl, and the other is an unbranchedsaturated C₉-C₂₀ alkyl. In some embodiments, R₁ and R₃ are eachindependently selected from a group consisting of:

In various embodiments, R₂ is selected from a group consisting of:

In some embodiments, R may be as described in International Pat. Pub.No. WO2019/152848 A1, which is incorporated herein by reference in itsentirety.

In some embodiments, an ionizable lipid is a compound of Formula (1-1)or Formula (1-2):

wherein n, R₁, R₂, and R₃ are as defined in Formula (1).

Preparation methods for the above compounds and compositions aredescribed herein below and/or known in the art.

It will be appreciated by those skilled in the art that in the processdescribed herein the functional groups of intermediate compounds mayneed to be protected by suitable protecting groups. Such functionalgroups include, e.g., hydroxyl, amino, mercapto, and carboxylic acid.Suitable protecting groups for hydroxyl include, e.g., trialkylsilyl ordiarylalkylsilyl (for example, t-butyldimethylsilyl,t-butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl, andthe like. Suitable protecting groups for amino, amidino, and guanidinoinclude, e.g., t-butoxycarbonyl, benzyloxycarbonyl, and the like.Suitable protecting groups for mercapto include, e.g., —C(O)—R″ (whereR″ is alkyl, aryl, or arylalkyl), p-methoxybenzyl, trityl, and the like.Suitable protecting groups for carboxylic acid include, e.g., alkyl,aryl, or arylalkyl esters. Protecting groups may be added or removed inaccordance with standard techniques, which are known to one skilled inthe art and as described herein. The use of protecting groups isdescribed in detail in, e.g., Green, T. W. and P. G. M. Wutz, ProtectiveGroups in Organic Synthesis (1999), 3rd Ed., Wiley. As one of skill inthe art would appreciate, the protecting group may also be a polymerresin such as a Wang resin, Rink resin, or a 2-chlorotrityl-chlorideresin.

It will also be appreciated by those skilled in the art, although suchprotected derivatives of compounds of this invention may not possesspharmacological activity as such, they may be administered to a mammaland thereafter metabolized in the body to form compounds of theinvention which are pharmacologically active. Such derivatives maytherefore be described as prodrugs. All prodrugs of compounds of thisinvention are included within the scope of the invention.

Furthermore, all compounds of the invention which exist in free base oracid form can be converted to their pharmaceutically acceptable salts bytreatment with the appropriate inorganic or organic base or acid bymethods known to one skilled in the art. Salts of the compounds of theinvention can also be converted to their free base or acid form bystandard techniques.

The following reaction scheme illustrates an exemplary method to makecompounds of Formula (1):

A1 are purchased or prepared according to methods known in the art.Reaction of A1 with diol A2 under appropriate condensation conditions(e.g., DCC) yields ester/alcohol A3, which can then be oxidized (e.g.,with PCC) to aldehyde A4. Reaction of A4 with amine A5 under reductiveamination conditions yields a compound of Formula (1).

The following reaction scheme illustrates a second exemplary method tomake compounds of Formula (1), wherein R₁ and R₃ are the same:

Modifications to the above reaction scheme, such as using protectinggroups, may yield compounds wherein R₁ and R₃ are different. The use ofprotecting groups, as well as other modification methods, to the abovereaction scheme will be readily apparent to one of ordinary skill in theart.

It is understood that one skilled in the art may be able to make thesecompounds by similar methods or by combining other methods known to oneskilled in the art. It is also understood that one skilled in the artwould be able to make other compounds of Formula (1) not specificallyillustrated herein by using the appropriate starting materials andmodifying the parameters of the synthesis. In general, startingmaterials may be obtained from sources such as Sigma Aldrich, LancasterSynthesis, Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA,etc. or synthesized according to sources known to those skilled in theart (see, e.g., Advanced Organic Chemistry: Reactions, Mechanisms, andStructure, 5th edition (Wiley, December 2000)) or prepared as describedin this invention.

In some embodiments, an ionizable lipid is a compound of Formula (2):

wherein each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, or 15.

In some embodiments, as used in Formula (2), R₁ and R₂ are as defined inFormula (1).

In some embodiments, as used in Formula (2), R₁ and R₂ are eachindependently selected from a group consisting of:

In some embodiments, R₁ and R₂ as use in Formula (2) may be as describedin International Pat. Pub. No. WO2011/095340 A1, which is incorporatedherein by reference in its entirety. In some embodiments, R₁ as used inFormula (2) may be as described in International Pat. Pub. No.WO2019/152557 A1, which is incorporated herein by reference in itsentirety.

In some embodiments, as used in Formula (2), R₃ is selected from a groupconsisting of:

In some embodiments, an ionizable lipid is a compound of Formula (3)

wherein X is selected from —O—, —S—, or —OC(O)—*, wherein * indicatesthe attachment point to R₁.

In some embodiments, an ionizable lipid is a compound of Formula (3-1):

In some embodiments, an ionizable lipid is a compound of Formula (3-2):

In some embodiments, an ionizable lipid is a compound of Formula (3-3):

In some embodiments, as used in Formula (3-1), (3-2), or (3-3), each R₁is independently a branched saturated C₉-C₂₀ alkyl. In some embodiments,each R₁ is independently selected from a group consisting of:

In some embodiments, each R₁ in Formula (3-1), (3-2), or (3-3) are thesame.

In some embodiments, as used in Formula (3-1), (3-2), or (3-3), R₂ isselected from a group consisting of:

In some embodiments, R₂ as used in Formula (3-1), (3-2), or (3-3) may beas described in International Pat. Pub. No. WO2019/152848A1, which isincorporated herein by reference in its entirety.

In some embodiments, an ionizable lipid is a compound of Formula (5):

wherein:

-   -   each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,        13, 14, or 15; and    -   R₂ is as defined in Formula (1).

In some embodiments, as used in Formula (5), R₄ and R₅ are defined as R₁and R₃, respectively, in Formula (1). In some embodiments, as used inFormula (5), R₄ and R₅ may be as described in International Pat. Pub.No. WO2019/191780 A1, which is incorporated herein by reference in itsentirety.

In some embodiments, an ionizable lipid of the disclosure is selectedfrom Table 10a. In some embodiments, the ionizable lipid is Lipid 26 inTable 10a. In some embodiments, the ionizable lipid is Lipid 27 in Table10a. In some embodiments, the ionizable lipid is Lipid 53 in Table 10a.In some embodiments, the ionizable lipid is Lipid 54 in Table 10a.

In some embodiments, an ionizable lipid of the disclosure is selectedfrom the group consisting of:

TABLE 10a Ioniz- able lipid num- ber Structure  1

 2

 3

 4

 5

 6

 7

 8

 9

 10

 11

 12

 13

 14

 15

 16

 17

 18

 19

 20

 21

 22

 23

 24

 25

 26

 27

 28

 29

 30

 31

 32

 33

 34

 35

 36

 37

 38

 39

 40

 41

 42

 43

 44

 45

 46

 47

 48

 49

 50

 51

 52

 53

 54

 55

 56

 57

 58

 59

 60

 61

 62

 63

 64

 65

 66

 67

 68

 69

 70

 71

 72

 73

 74

 75

 76

 77

 78

 79

 80

 81

 82

 83

 84

 85

 86

 87

 88

 89

 90

 91

 92

 93

 94

 95

 96

 97

 98

 99

100

101

102

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125

126

127

128

129

130

131

132

133

134

135

136

In some embodiments, the ionizable lipid has a beta-hydroxyl amine headgroup. In some embodiments, the ionizable lipid has a gamma-hydroxylamine head group.

In some embodiments, an ionizable lipid of the disclosure is a lipidselected from Table 10b. In some embodiments, an ionizable lipid of thedisclosure is Lipid 15 from Table 10b. In an embodiment, the ionizablelipid is described in US patent publication number US20170210697A1. Inan embodiment, the ionizable lipid is described in US patent publicationnumber US20170119904A1.

TABLE 10b Ionizable lipid number Structure 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

In some embodiments, an ionizable lipid has one of the structures setforth in Table 10 below.

TABLE 10 Num- ber Structure 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

In some embodiments, the ionizable lipid has one of the structures setforth in Table 11 below. In some embodiments, the ionizable lipid as setforth in Table 11 is as described in international patent applicationPCT/US2010/061058.

TABLE 11

meso, cis

Racemic, cis

Racemic, trans

Racemic, trans

Racemic cis

cis Racemic and optically pure

Racemic and optically pure

cis Racemic and optically pure

cis Racemic and optically pure

cis Racemic and optically pure

meso compound

racemic compound

racemic compound

racemic compound

cis Racemic and optically pure

meso compound

racemic compound

racemic compound

n = 1-10

n = 0-6

n = 0-6

Racemic and optically pure

n = 0-6

n = 0-6

cis Racemic and optically pure

n = 0-6

Racemic and optically pure

n = 0-6

n = 0-6

n = 0-6

n = 0-6

n = 0-6

Q is O, NH, NMe

Q is O, NH, NMe

Q is O, NH, NMe

Q is O, NH, NMe

Q is O, NH, NMe

Q is O, NH, NMe

Q is O, NH, NMe

Q is O, NH, NMe

Q is O, NH, NMe

Q is O, NH, NMe

Q is O, NH, NMe

Q is O, NH, NMe

Q is O, NH, NMe

Q is O, NH, NMe

Q is O, NH, NMe

Q is O, NH, NMe

Q is O, NH, NMe

Q is O, NH, NMe

Q is O, NH, NMe

Q is O, NH, NMe

Q is O, NH, NMe

Q is O, NH, NMe

Q is O, NH, NMe

Q is O, NH, NMe

Q is O, NH, NMe

Q is O, NH, NMe

Q is O, NH, NMe

Q is O, NH, NMe

Q is O, NH, NMe

Q is O, NH, NMe

Q is O, NH, NMe

X = O, S, NH, NMe; n = 0-6

X = O, S, NH, NMe; n = 0-6

n = 0-6

Racemic and optically pure

n = 1-10

Racemic and optically pure

n = 0-6

Q is O

Q is O

Q is O

Q is O

Q is O

Q is O

Q is O

Q is O

Q is O

Q is O

Q is O

Q is O

Q is O

Q is O

Q is O

Q is O

Q is O

Q is O

Q is O

Q is O

Q is O

Q is O

Q is O

X = O, S, NH, NE; n = 0-6

X = O, S, NH, NE; n = 0-6

e.g., X = Me, OH, Cl, etc

n = 0-6

n = 0-6

n = 0-6

n = 0-6

n = 0-6

n = 0-6

n = 0-6

Q is NH, NMe

Q is NH, NMe

Q is NH, NMe

Q is NH, NMe

Q is NH, NMe

Q is NH, NMe

Q is NH, NMe

Q is NH, NMe

Q is NH, NMe

Q is NH, NMe

Q is NH, NMe

Q is NH, NMe

Q is NH, NMe

Q is NH, NMe

Q is NH, NMe

Q is NH, NMe

Q is NH, NMe

Q is NH, NMe

Q is NH, NMe

Q is NH, NMe

Q is NH, NMe

Q is NH, NMe

Q is NH, NMe

Q is NH, NMe

In some embodiments, the transfer vehicle comprises Lipid A, Lipid B,Lipid C, and/or Lipid D. In some embodiments, inclusion of Lipid A,Lipid B, Lipid C, and/or Lipid D improves encapsulation and/or endosomalescape. In some embodiments, Lipid A, Lipid B, Lipid C, and/or Lipid Dare described in international patent application PCT/US2017/028981.

In some embodiments, an ionizable lipid is Lipid A, which is(9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyloctadeca9,12-dienoate, also called3-((4,44bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate. Lipid A can be depicted as:

Lipid A may be synthesized according to WO2015/095340 (e.g., pp. 84-86),incorporated by reference in its entirety.

In some embodiments, an ionizable lipid is Lipid B, which is((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(octane-8,1-diyl)bis(decanoate).Lipid B can be depicted as:

Lipid B may be synthesized according to WO2014/136086 (e.g., pp.107-09), incorporated by reference in its entirety.

In some embodiments, an ionizable lipid is Lipid C, which is2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diyl(9Z,9′Z,12Z,12′Z)-bis(octadeca-9,12-dienoate).Lipid C can be depicted as:

In some embodiments, an ionizable lipid is Lipid D, which is3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl3-octylundecanoate. Lipid D can be depicted as:

Lipid C and Lipid D may be synthesized according to WO2015/095340,incorporated by reference in its entirety.

In some embodiments, an ionizable lipid is described in US patentpublication number 20190321489. In some embodiments, an ionizable lipidis described in international patent publication WO 2010/053572,incorporated herein by reference. In some embodiments, an ionizablelipid is C12-200, described at paragraph [00225] of WO 2010/053572.

Several ionizable lipids have been described in the literature, many ofwhich are commercially available. In certain embodiments, such ionizablelipids are included in the transfer vehicles described herein. In someembodiments, the ionizable lipidN-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride or “DOTMA”is used. (Felgner et al. Proc. Nat'l Acad. Sci. 84, 7413 (1987); U.S.Pat. No. 4,897,355). DOTMA can be formulated alone or can be combinedwith a neutral lipid, dioleoylphosphatidylethanolamine or “DOPE” orother cationic or non-cationic lipids into a lipid nanoparticle. Othersuitable cationic lipids include, for example, ionizable cationic lipidsas described in U.S. provisional patent application 61/617,468, filedMar. 29, 2012 (incorporated herein by reference), such as, e.g.,(15Z,18Z)—N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine(HGT5000),(15Z,18Z)—N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-trien-1-amine(HGT5001), and(15Z,18Z)—N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-5,15,18-trien-1-amine(HGT5002), C12-200 (described in WO 2010/053572),2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine(DLinKC2-DMA)) (See, WO 2010/042877; Semple et al., Nature Biotech.28:172-176 (2010)),2-(2,2-di((9Z,2Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine(DLin-KC2-DMA),(3S,10R,13R,17R)-10,13-dimethyl-174(R)-6-methylheptan-2-yl)-2, 3, 4, 7,8, 9, 10, 11, 12, 13, 14, 15, 16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl3-(1H-imidazol-4-yl)propanoate (ICE),(15Z,18Z)—N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine(HGT5000),(15Z,18Z)—N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-trien-1-amine(HGT5001), (15Z,18Z)—N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-5,15,18-trien-1-amine(HGT5002), 5-carboxyspermylglycine-dioctadecylamide (DOGS),2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium(DOSPA) (Behr et al. Proc. Nat.'l Acad. Sci. 86, 6982 (1989); U.S. Pat.Nos. 5,171,678; 5,334,761), 1,2-Dioleoyl-3-Dimethylammonium-Propane(DODAP), 1,2-Dioleoyl-3-Trimethylammonium-Propane or (DOTAP).Contemplated ionizable lipids also include1,2-distcaryloxy-N,N-dimethyl-3-aminopropane (DS DMA),1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA),1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA),1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA),N-dioleyl-N,N-dimethylammonium chloride (DODAC),N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (DMRIE),3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane(CLinDMA),2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethyl-1-(cis,cis-9′-octadecadienoxy)propane(CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA),1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP),2,3-Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP),1,2-N,N′-Dilinoleylcarbamyl-3-dimethylamninopropane (DLincarbDAP),1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP),2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA) orGL67, or mixtures thereof. (Heyes, J., et al., J Controlled Release 107:276-287 (2005); Morrissey, D V., et al., Nat. Biotechnol. 23(8):1003-1007 (2005); PCT Publication WO2005/121348A1). The use ofcholesterol-based ionizable lipids to formulate the transfer vehicles(e.g., lipid nanoparticles) is also contemplated by the presentinvention. Such cholesterol-based ionizable lipids can be used, eitheralone or in combination with other lipids. Suitable cholesterol-basedionizable lipids include, for example, DC-Cholesterol(N,N-dimethyl-N-ethylcarboxamidocholesterol), and1,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al., Biochem. Biophys.Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997);U.S. Pat. No. 5,744,335).

Also contemplated are cationic lipids such as dialkylamino-based,imidazole-based, and guanidinium-based lipids. For example, alsocontemplated is the use of the ionizable lipid (3S,10R, 13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10,11, 12, 13, 14, 15, 16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl3-(1H-imidazol-4-yl)propanoate (ICE), as disclosed in InternationalApplication No. PCT/US2010/058457, incorporated herein by reference.

Also contemplated are ionizable lipids such as the dialkylamino-based,imidazole-based, and guanidinium-based lipids. For example, certainembodiments are directed to a composition comprising one or moreimidazole-based ionizable lipids, for example, the imidazole cholesterolester or “ICE” lipid, (3S, 10R, 13R, 17R)-10,13-dimethyl-174(R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl3-(1H-imidazol-4-yl)propanoate, as represented by structure (XIII)below. In an embodiment, a transfer vehicle for delivery of circRNA maycomprise one or more imidazole-based ionizable lipids, for example, theimidazole cholesterol ester or “ICE” lipid (3S, 10R, 13R, 17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 9, 10, 11, 12, 13,14, 15, 16, 17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl3-(1H-imidazol-4-yl)propanoate, as represented by structure (XIII).

Without wishing to be bound by a particular theory, it is believed thatthe fusogenicity of the imidazole-based cationic lipid ICE is related tothe endosomal disruption which is facilitated by the imidazole group,which has a lower pKa relative to traditional ionizable lipids. Theendosomal disruption in turn promotes osmotic swelling and thedisruption of the liposomal membrane, followed by the transfection orintracellular release of the nucleic acid(s) contents loaded thereininto the target cell.

The imidazole-based ionizable lipids are also characterized by theirreduced toxicity relative to other ionizable lipids.

In some embodiments, an ionizable lipid is described by US patentpublication number 20190314284. In certain embodiments, the an ionizablelipid is described by structure 3, 4, 5, 6, 7, 8, 9, or 10 (e.g.,HGT4001, HGT4002, HGT4003, HGT4004 and/or HGT4005). In certainembodiments, the one or more cleavable functional groups (e.g., adisulfide) allow, for example, a hydrophilic functional head-group todissociate from a lipophilic functional tail-group of the compound(e.g., upon exposure to oxidative, reducing or acidic conditions),thereby facilitating a phase transition in the lipid bilayer of the oneor more target cells. For example, when a transfer vehicle (e.g., alipid nanoparticle) comprises one or more of the lipids of structures3-10, the phase transition in the lipid bilayer of the one or moretarget cells facilitates the delivery of the circRNA into the one ormore target cells.

In certain embodiments, the ionizable lipid is described by structure(XIV),

wherein:

R₁ is selected from the group consisting of imidazole, guanidinium,amino, imine, enamine, an optionally-substituted alkyl amino (e.g., analkyl amino such as dimethylamino) and pyridyl;

R₂ is selected from the group consisting of structure XV and structureXVI;

wherein R₃ and R₄ are each independently selected from the groupconsisting of an optionally substituted, variably saturated orunsaturated C₆-C₂₀ alkyl and an optionally substituted, variablysaturated or unsaturated C₆-C₂₀ acyl; and wherein n is zero or anypositive integer (e.g., one, two, three, four, five, six, seven, eight,nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,seventeen, eighteen, nineteen, twenty or more). In certain embodiments,R₃ and R₄ are each an optionally substituted, polyunsaturated C₁₈ alkyl,while in other embodiments R₃ and R₄ are each an unsubstituted,polyunsaturated C₁₈ alkyl. In certain embodiments, one or more of R₃ andR₄ are (9Z,12Z)-octadeca-9,12-dien.

Also disclosed herein are pharmaceutical compositions that comprise thecompound of structure XIV, wherein R₁ is selected from the groupconsisting of imidazole, guanidinium, amino, imine, enamine, anoptionally-substituted alkyl amino (e.g., an alkyl amino such asdimethylamino) and pyridyl; wherein R₂ is structure XV; and wherein n iszero or any positive integer. Further disclosed herein arepharmaceutical compositions comprising the compound of structure XIV,wherein R₁ is selected from the group consisting of imidazole,guanidinium, amino, imine, enamine, an optionally-substituted alkylamino (e.g., an alkyl amino such as dimethylamino) and pyridyl; whereinR₂ is structure XVI; wherein R₃ and R₄ are each independently selectedfrom the group consisting of an optionally substituted, variablysaturated or unsaturated C₆-C₂₀ alkyl and an optionally substituted,variably saturated or unsaturated C₆-C₂₀ acyl; and wherein n is zero orany positive integer. In certain embodiments. R₃ and R₄ are each anoptionally substituted, polyunsaturated Cis alkyl, while in otherembodiments R₃ and R₄ are each an unsubstituted, polyunsaturated C₁₈alkyl (e.g., octadeca-9,12-dien).

In certain embodiments, the R₁ group or head-group is a polar orhydrophilic group (e.g., one or more of the imidazole, guanidinium andamino groups) and is bound to the R₂ lipid group by way of the disulfide(S—S) cleavable linker group, for example as depicted in structure XIV.Other contemplated cleavable linker groups may include compositions thatcomprise one or more disulfide (S—S) linker group bound (e.g.,covalently bound) to, for example an alkyl group (e.g., C₁ to C₁₀alkyl). In certain embodiments, the R₁ group is covalently bound to thecleavable linker group by way of a C₁-C₂₀ alkyl group (e.g., where n isone to twenty), or alternatively may be directly bound to the cleavablelinker group (e.g., where n is zero). In certain embodiments, thedisulfide linker group is cleavable in vitro and/or in vivo (e.g.,enzymatically cleavable or cleavable upon exposure to acidic or reducingconditions).

In certain embodiments, the inventions relate to the compound5-(((10,13-dimethyl-17-(6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-Cyclopenta[a]phenanthren-3-yl)disulfanyl)methyl)-1H-imidazole,having structure XVII (referred to herein as “HGT4001”).

In certain embodiments, the inventions relate to the compound1-(2-(((3S,10R,13R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)disulfanyl)ethyl)guanidine,having structure XVIII (referred to herein as “HGT4002”).

In certain embodiments, the inventions relate to the compound2-((2,3-Bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)propyl)disulfanyl)-N,N-dimethylethanamine,having structure XIX (referred to herein as “HGT4003”).

In other embodiments, the inventions relate to the compound5-(((2,3-bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)propyl)disulfanyl)methyl)-1H-imidazolehaving the structure of structure XX (referred to herein as “HGT4004”).

In still other embodiments, the inventions relate to the compound1-(((2,3-bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)propyl)disulfanyl)methyl)guanidinehaving structure XXI (referred to herein as “HGT4005”).

In certain embodiments, the compounds described as structures 3-10 areionizable lipids.

The compounds, and in particular the imidazole-based compounds describedas structures 3-8 (e.g., HGT4001 and HGT4004), are characterized bytheir reduced toxicity, in particular relative to traditional ionizablelipids. In some embodiments, the transfer vehicles described hereincomprise one or more imidazole-based ionizable lipid compounds such thatthe relative concentration of other more toxic ionizable lipids in suchpharmaceutical or liposomal composition may be reduced or otherwiseeliminated.

The ionizable lipids include those disclosed in international patentapplication PCT/US2019/025246, and US patent publications 2017/0190661and 2017/0114010, incorporated herein by reference in their entirety.The ionizable lipids may include a lipid selected from the followingtables 12, 13, 14, or 15.

TABLE 12

ATX-001

ATX-002

ATX-003

ATX-004

ATX-005

ATX-006

ATX-007

ATX-008

ATX-009

ATX-010

ATX-011

ATX-012

ATX-013

ATX-014

ATX-015

ATX-016

ATX-017

ATX-018

ATX-019

ATX-020

ATX-021

ATX-022

ATX-023

ATX-024

ATX-025

ATX-026

ATX-027

ATX-028

ATX-029

ATX-030

ATX-031

ATX-032

TABLE 13

ATX-B-1

ATX-B-2

ATX-B-3

ATX-B-4

ATX-B-5

ATX-B-6

ATX-B-7

ATX-B-8

ATX-B-9

ATX-B-10

ATX-B-11

ATX-B-12

TABLE 14 Compound ATX-#

0063

0130

0131

0044

0111

0132

0134

0133

0064

0061

0100

0117

0114

0115

0101

0106

0116

0043

0086

0058

0081

0123

0122

0057

0088

0087

0124

0128

0127

0126

0129

0082

0085

0083

0121

0091

0102

0098

0092

0084

0095

0125

0094

0109

0110

0118

0108

0107

0093

0097

0096

TABLE 15

11

13

14

15

16

17

18

19

20

In some embodiments, an ionizable lipid is as described in internationalpatent application PCT/US2019/015913. In some embodiments, an ionizablelipid is chosen from the following:

5.1 Amine Lipids

In certain embodiments, transfer vehicle compositions for the deliveryof circular RNA comprise an amine lipid. In certain embodiments, anionizable lipid is an amine lipid. In some embodiments, an amine lipidis described in international patent application PCT/US2018/053569.

In some embodiments, the amine lipid is Lipid E, which is (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyloctadeca-9, 12-dienoate.

Lipid E can be depicted as:

Lipid E may be synthesized according to WO2015/095340 (e.g., pp. 84-86).In certain embodiments, the amine lipid is an equivalent to Lipid E.

In certain embodiments, an amine lipid is an analog of Lipid E. Incertain embodiments, a Lipid E analog is an acetal analog of Lipid E. Inparticular transfer vehicle compositions, the acetal analog is a C4-C12acetal analog. In some embodiments, the acetal analog is a C5-C12 acetalanalog. In additional embodiments, the acetal analog is a C5-C10 acetalanalog. In further embodiments, the acetal analog is chosen from a C4,C5, C6, C7, C9, C10, C11 and C12 acetal analog.

Amine lipids and other biodegradable lipids suitable for use in thetransfer vehicles, e.g., lipid nanoparticles, described herein arebiodegradable in vivo. The amine lipids described herein have lowtoxicity (e.g., are tolerated in animal models without adverse effect inamounts of greater than or equal to 10 mg/kg). In certain embodiments,transfer vehicles composing an amine lipid include those where at least75% of the amine lipid is cleared from the plasma within 8, 10, 12, 24,or 48 hours, or 3, 4, 5, 6, 7, or 10 days.

Biodegradable lipids include, for example, the biodegradable lipids ofWO2017/173054, WO2015/095340, and WO2014/136086.

Lipid clearance may be measured by methods known by persons of skill inthe art. See, for example, Maier, M. A., et al. Biodegradable LipidsEnabling Rapidly Eliminated Lipid Nanoparticles for Systemic Delivery ofRNAi Therapeutics. Mol. Ther. 2013, 21(8), 1570-78.

Transfer vehicle compositions comprising an amine lipid can lead to anincreased clearance rate. In some embodiments, the clearance rate is alipid clearance rate, for example the rate at which a lipid is clearedfrom the blood, serum, or plasma. In some embodiments, the clearancerate is an RNA clearance rate, for example the rate at which an circRNAis cleared from the blood, serum, or plasma. In some embodiments, theclearance rate is the rate at which transfer vehicles are cleared fromthe blood, serum, or plasma. In some embodiments, the clearance rate isthe rate at which transfer vehicles are cleared from a tissue, such asliver tissue or spleen tissue. In certain embodiments, a high rate ofclearance leads to a safety profile with no substantial adverse effects.The amine lipids and biodegradable lipids may reduce transfer vehicleaccumulation in circulation and in tissues. In some embodiments, areduction in transfer vehicle accumulation in circulation and in tissuesleads to a safety profile with no substantial adverse effects.

Lipids may be ionizable depending upon the pH of the medium they are in.For example, in a slightly acidic medium, the lipid, such as an aminelipid, may be protonated and thus bear a positive charge. Conversely, ina slightly basic medium, such as, for example, blood, where pH isapproximately 7.35, the lipid, such as an amine lipid, may not beprotonated and thus bear no charge.

The ability of a lipid to bear a charge is related to its intrinsic pKa.In some embodiments, the amine lipids of the present disclosure mayeach, independently, have a pKa in the range of from about 5.1 to about7.4. In some embodiments, the bioavailable lipids of the presentdisclosure may each, independently, have a pKa in the range of fromabout 5.1 to about 7.4. For example, the amine lipids of the presentdisclosure may each, independently, have a pKa in the range of fromabout 5.8 to about 6.5. Lipids with a pKa ranging from about 5.1 toabout 7.4 are effective for delivery of cargo in vivo, e.g., to theliver. Further, it has been found that lipids with a pKa ranging fromabout 5.3 to about 6.4 are effective for delivery in vivo, e.g., intotumors. See, e.g., WO2014/136086.

5.2 Lipids Containing a Disulfide Bond

In some embodiments, the ionizable lipid is described in U.S. Pat. No.9,708,628.

The present invention provides a lipid represented by structure (XXII):

In structure (XXII), X^(a) and X^(b) are each independently X¹ or X²shown below.

R⁴ in X¹ is an alkyl group having 1-6 carbon atoms, which may be linear,branched or cyclic. The alkyl group preferably has a carbon number of1-3. Specific examples of the alkyl group having 1-6 carbon atomsinclude methyl group, ethyl group, propyl group, isopropyl group,n-butyl group, sec-butyl group, isobutyl group, tert-butyl group, pentylgroup, isopentyl group, neopentyl group, t-pentyl group,1,2-dimethylpropyl group, 2-methylbutyl group, 2-methylpentyl group,3-methylpentyl group, 2,2-dimethylbutyl group, 2,3-dimethylbutyl group,cyclohexyl group and the like. R⁴ is preferably a methyl group, an ethylgroup, a propyl group or an isopropyl group, most preferably a methylgroup.

The s in X² is 1 or 2. When s is 1, X² is a pyrrolidinium group, andwhen s is 2, X² is a piperidinium group. s is preferably 2. While thebinding direction of X² is not limited, a nitrogen atom in X² preferablybinds to R^(1a) and R^(1b).

X^(a) may be the same as or different from X^(b), and X^(a) ispreferably the same group as X^(b).

n^(a) and n^(b) are each independently 0 or 1, preferably 1. When n^(a)is 1, R^(1a) binds to X^(a) via Y^(a) and R^(2a), and when n^(a) is 0, astructure of R^(3a)—X^(a)—R^(1a)—S— is taken. Similarly, when n^(b) is1, R^(3b) binds to X^(b) via Y^(b) and R^(2b), and when n^(b) is 0, astructure of R^(3b)—X^(b)—R^(1b)—S— is taken.

n^(a) may be the same as or different from n^(b), and n^(a) ispreferably the same as n^(b).

R^(1a) and R^(1b) are each independently an alkylene group having 1-6carbon atoms, which may be linear or branched, preferably linear.Specific examples of the alkylene group having 1-6 carbon atoms includemethylene group, ethylene group, trimethylene group, isopropylene group,tetramethylene group, isobutylene group, pentamethylene group,neopentylene group and the like. R^(1a) and R^(1b) are each preferably amethylene group, an ethylene group, a trimethylene group, anisopropylene group or a tetramethylene group, most preferably anethylene group.

R^(1a) may be the same as or different from R^(1b), and R^(1a) ispreferably the same group as R^(1b).

R^(2a) and R^(2b) are each independently an alkylene group having 1-6carbon atoms, which may be linear or branched, preferably linear.Examples of the alkylene group having 1-6 carbon atoms include thoserecited as the examples of the alkylene group having 1-6 carbon atomsfor R^(1a) or R_(1b). R^(2a) and R_(2b) are each preferably a methylenegroup, an ethylene group, a trimethylene group, an isopropylene group ora tetramethylene group.

When X^(a) and X^(b) are each X¹, R^(2a) and R^(2b) are preferablytrimethylene groups. When X^(a) and X^(b) are each X², R^(2a) and R^(2b)are preferably ethylene groups.

R^(2a) may be the same as or different from R^(2b), and R^(2a) ispreferably the same group as R^(2b).

Y^(a) and Y^(b) are each independently an ester bond, an amide bond, acarbamate bond, an ether bond or a urea bond, preferably an ester bond,an amide bond or a carbamate bond, most preferably an ester bond. Whilethe binding direction of Y^(a) and Y^(b) is not limited, when Y^(a) isan ester bond, a structure of R^(3a)—CO—O—R^(2a)— is preferable, andwhen Y^(b) is an ester bond, a structure of R^(3b)—CO—O—R^(2b)— ispreferable.

Y^(a) may be the same as or different from Y^(b), and Y^(a) ispreferably the same group as Y^(b).

R^(1a) and R^(3b) are each independently a sterol residue, a liposolublevitamin residue or an aliphatic hydrocarbon group having 12-22 carbonatoms, preferably a liposoluble vitamin residue or an aliphatichydrocarbon group having 12-22 carbon atoms, most preferably aliposoluble vitamin residue.

Examples of the sterol residue include a cholesteryl group (cholesterolresidue), a cholestaryl group (cholestanol residue), a stigmasterylgroup (stigmasterol residue), a β-sitosteryl group (β-sitosterolresidue), a lanosteryl group (lanosterol residue), and an ergosterylgroup (ergosterol residue) and the like. The sterol residue ispreferably a cholesteryl group or a cholestaryl group.

As the liposoluble vitamin residue, a residue derived from liposolublevitamin, as well as a residue derived from a derivative obtained byappropriately converting a hydroxyl group, aldehyde or carboxylic acid,which is a functional group in liposoluble vitamin, to other reactivefunctional group can be used. As for liposoluble vitamin having ahydroxyl group, for example, the hydroxyl group can be converted to acarboxylic acid by reacting with succinic acid anhydride, glutaric acidanhydride and the like. Examples of the liposoluble vitamin includeretinoic acid, retinol, retinal, ergosterol, 7-dehydrocholesterol,calciferol, cholecalciferol, dihydroergocalciferol, dihydrotachysterol,tocopherol, tocotrienol and the like. Preferable examples of theliposoluble vitamin include retinoic acid and tocopherol.

The aliphatic hydrocarbon group having 12-22 carbon atoms may be linearor branched, preferably linear. The aliphatic hydrocarbon group may besaturated or unsaturated. In the case of an unsaturated aliphatichydrocarbon group, the aliphatic hydrocarbon group generally contains1-6, preferably 1-3, more preferably 1-2 unsaturated bonds. While theunsaturated bond includes a carbon-carbon double bond and acarbon-carbon triple bond, it is preferably a carbon-carbon double bond.The aliphatic hydrocarbon group has a carbon number of preferably 12-18,most preferably 13-17. While the aliphatic hydrocarbon group includes analkyl group, an alkenyl group, an alkynyl group and the like, it ispreferably an alkyl group or an alkenyl group. Specific examples of thealiphatic hydrocarbon group having 12-22 carbon atoms include dodecylgroup, tridecyl group, tetradecyl group, pentadecyl group, hexadecylgroup, heptadecyl group, octadecyl group, nonadecyl group, icosyl group,henicosyl group, docosyl group, dodecenyl group, tridecenyl group,tetradecenyl group, pentadecenyl group, hexadecenyl group, heptadecenylgroup, octadecenyl group, nonadecenyl group, icosenyl group, henicosenylgroup, docosenyl group, decadienyl group, tridecadienyl group,tetradecadienyl group, pentadecadienyl group, hexadecadienyl group,heptadecadienyl group, octadecadienyl group, nonadecadienyl group,icosadienyl group, henicosadienyl group, docosadienyl group,octadecatrienyl group, icosatrienyl group, icosatetraenyl group,icosapentaenyl group, docosahexaenyl group, isostearyl group and thelike. The aliphatic hydrocarbon group having 12-22 carbon atoms ispreferably tridecyl group, tetradecyl group, heptadecyl group, octadecylgroup, heptadecadienyl group or octadecadienyl group, particularlypreferably tridecyl group, heptadecyl group or heptadecadienyl group.

In one embodiment, an aliphatic hydrocarbon group having 12-22 carbonatoms, which is derived from fatty acid, aliphatic alcohol, or aliphaticamine is used. When R^(1a) (or R^(3b)) is derived from fatty acid, Y^(a)(or Y^(b)) is an ester bond or an amide bond, and fatty acid-derivedcarbonyl carbon is included in Y^(a) (or Y^(b)). For example, whenlinoleic acid is used, R^(1a) (or R^(3b)) is a heptadecadienyl group.

R^(1a) may be the same as or different from R^(3b), and R^(1a) ispreferably the same group as R^(3b).

In one embodiment, X^(a) is the same as X^(b), n^(a) is the same asn^(b), R^(1a) is the same as R^(1b), R^(2a) is the same as R^(2b),R^(3a) is the same as R^(3b), and Y^(a) is the same as Y^(b).

In one embodiment,

-   -   X^(a) and X^(b) are each independently X1,    -   R⁴ is an alkyl group having 1-3 carbon atoms, n^(a) and n^(b)        are each 1,    -   R^(1a) and R^(1b) are each independently an alkylene group        having 1-6 carbon atoms,    -   R^(1a) and R^(2b) are each independently an alkylene group        having 1-6 carbon atoms,    -   Y^(a) and Y^(b) are each an ester bond or an amide bond, and    -   R^(3a) and R^(3b) are each independently an aliphatic        hydrocarbon group having 12-22 carbon atoms.

In one embodiment,

-   -   X^(a) and X^(b) are each X1,    -   R⁴ is an alkyl group having 1-3 carbon atoms, n^(a) and n^(b)        are each 1,    -   R^(1a) and R^(1b) are each an alkylene group having 1-6 carbon        atoms,    -   R^(2a) and R^(2b) are each an alkylene group having 1-6 carbon        atoms,    -   Y^(a) and Y^(b) are each an ester bond or an amide bond,    -   R^(3a) and R^(3b) are each an aliphatic hydrocarbon group having        12-22 carbon atoms,    -   X^(a) is the same as X^(b),    -   R^(1a) is the same as R^(1b),    -   R^(2a) is the same as R^(2b), and    -   R^(3a) is the same as R^(3b).

In one embodiment,

-   -   X^(a) and X^(b) are each X¹,    -   R⁴ is a methyl group, n^(a) and n^(b) are each 1,    -   R^(1a) and R^(1b) are each an ethylene group,    -   R^(2a) and R^(2b) are each a trimethylene group,    -   Y^(a) and Y^(b) are each —CO—O—, and    -   R^(3a) and R^(3b) are each independently an alkyl group or        alkenyl group having 13-17 carbon atoms.

In one embodiment,

-   -   X^(a) and X^(b) are each X¹,    -   R⁴ is a methyl group, n^(a) and n^(b) are each 1,    -   R^(1a) and R^(1b) are each an ethylene group,    -   R^(2a) and R^(2b) are each a trimethylene group,    -   Y^(a) and Y^(b) are each —CO—O—,    -   R^(1a) and R^(3b) are each an alkyl group or alkenyl group        having 13-17 carbon atoms, and    -   R^(3a) is the same as R^(3b).

In one embodiment,

-   -   X^(a) and X^(b) are each independently X¹,    -   R⁴ is an alkyl group having 1-3 carbon atoms, n^(a) and n^(b)        are each 1,    -   R^(1a) and R^(1b) are each independently an alkylene group        having 1-6 carbon atoms,    -   R^(2a) and R^(2b) are each independently an alkylene group        having 1-6 carbon atoms,    -   Y^(a) and Y^(b) are each an ester bond or an amide bond, and    -   R^(3a) and R^(3b) are each independently a liposoluble vitamin        residue (e.g., retinoic acid residue, tocopherol residue).

In one embodiment,

-   -   X^(a) and X^(b) are each X¹,    -   R⁴ is an alkyl group having 1-3 carbon atoms, n^(a) and n^(b)        are each 1,    -   R^(1a) and R^(1b) are each an alkylene group having 1-6 carbon        atoms,    -   R^(2a) and R^(2b) are each an alkylene group having 1-6 carbon        atoms,    -   Y^(a) and Y^(b) are each an ester bond or an amide bond,    -   R^(3a) and R^(3b) are each a liposoluble vitamin residue (e.g.,        retinoic acid residue, tocopherol residue),    -   X^(a) is the same as X^(b),    -   R^(1a) is the same as R^(1b),    -   R^(2a) is the same as R^(2b), and    -   R^(1a) is the same as R^(3b).

In one embodiment,

-   -   X^(a) and X^(b) are each X¹,    -   R⁴ is a methyl group, n^(a) and n^(b) are each 1,    -   R^(1a) and R^(1b) are each an ethylene group,    -   R^(2a) and R^(2b) are each a trimethylene group,    -   Y^(a) and Y^(b) are each —CO—O—, and    -   R^(3a) and R^(3b) are each independently a liposoluble vitamin        residue (e.g., retinoic acid residue, tocopherol residue).

In one embodiment,

-   -   X^(a) and X^(b) are each X¹,    -   R⁴ is a methyl group, n^(a) and n^(b) are each 1,    -   R^(1a) and R^(1b) are each an ethylene group,    -   R^(2a) and R^(2b) are each a trimethylene group,    -   Y^(a) and Y^(b) are each —CO—O—,    -   R^(3a) and R^(3b) are each a liposoluble vitamin residue (e.g.,        retinoic acid residue, tocopherol residue), and    -   R^(3a) is the same as R^(3b).

In one embodiment,

-   -   X^(a) and X^(b) are each independently X²,    -   t is 2,    -   R^(1a) and R^(1b) are each independently an alkylene group        having 1-6 carbon atoms,    -   R^(2a) and R^(2b) are each independently an alkylene group        having 1-6 carbon atoms,    -   Y^(a) and Y^(b) are each an ester bond, and    -   R^(3a) and R^(3b) are each independently a liposoluble vitamin        residue (e.g., retinoic acid residue, tocopherol residue) or an        aliphatic hydrocarbon group having 12-22 carbon atoms (e.g.,        alkyl group having 12-22 carbon atoms).

In one embodiment,

-   -   X^(a) and X^(b) are each independently X²,    -   t is 2,    -   R^(1a) and R^(1b) are each independently an alkylene group        having 1-6 carbon atoms,    -   R^(2a) and R^(2b) are each independently an alkylene group        having 1-6 carbon atoms,    -   Y^(a) and Y^(b) are each an ester bond,    -   R^(3a) and R^(3b) are each independently a liposoluble vitamin        residue (e.g., retinoic acid residue, tocopherol residue) or an        aliphatic hydrocarbon group having 12-22 carbon atoms (e.g.,        alkyl group having 12-22 carbon atoms),    -   X^(a) is the same as X^(b),    -   R^(1a) is the same as R^(1b),    -   R^(2a) is the same as R^(2b), and    -   R^(3a) is the same as R^(3b).

In one embodiment,

-   -   X^(a) and X^(b) are each independently X²,    -   t is 2,    -   R^(1a) and R^(1b) are each an ethylene group,    -   R^(2a) and R^(2b) are each independently an alkylene group        having 1-6 carbon atoms,    -   Y^(a) and Y^(b) are each an ester bond,    -   R^(3a) and R^(3b) are each independently a liposoluble vitamin        residue (e.g., retinoic acid residue, tocopherol residue) or an        aliphatic hydrocarbon group having 12-22 carbon atoms (e.g.,        alkyl group having 12-22 carbon atoms),    -   X^(a) is the same as X^(b),    -   R^(2a) is the same as R^(2b), and    -   R^(3a) is the same as R^(3b).

In some embodiments, an ionizable lipid has one of the structures setforth in Table 15b below.

TABLE 15b Num- ber Structure 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

A lipid of the present invention may have an —S—S-(disulfide) bond. Theproduction method for such a compound includes, for example, a methodincluding producingR^(1a)—(Y^(a)—R^(2a))n ^(a)-X^(a)—R^(1a)—SH, andR^(3b)—(Y^(b)—R^(2b))n ^(b)-X^(b)—R^(1b)—SH, andsubjecting them to oxidation (coupling) to give a compound containing—S—S—, a method including sequentially bonding necessary parts to acompound containing an —S—S— bond to finally obtain the compound of thepresent invention and the like. Preferred is the latter method.

A specific example of the latter method is shown below, which is not tobe construed as limiting.

Examples of the starting compound include —S—S— bond-containing twoterminal carboxylic acid, two terminal carboxylate, two terminal amine,two terminal isocyanate, two terminal alcohol, two terminal alcoholhaving a leaving group such as MsO (mesylate group) and the like, a twoterminal carbonate having a leaving group such as pNP(p-nitrophenylcarbonate group) and the like.

For example, when a compound containing X¹ or X² for X^(a) and X^(b) isproduced, two terminal functional groups of compound (1) containing an—S—S— bond are reacted with an —NH— group in compound (2) having the—NH— group and one functional group at the terminal, the functionalgroup at the terminal in compound (2) which did not contribute to thereaction is reacted with a functional group in compound (3) containingR³, whereby the compound of the present invention containing an —S—S—bond, R^(1a) and R^(1b), X^(a) and X^(b), R^(2a) and R_(2b), Y^(a) andY^(b), and R^(3a) and R^(3b) can be obtained.

In the reaction of compound (1) and compound (2), an alkali catalystsuch as potassium carbonate, sodium carbonate, potassium t-butoxide andthe like may be used as a catalyst, or the reaction may be performedwithout a catalyst. Preferably, potassium carbonate or sodium carbonateis used as a catalyst.

The amount of catalyst is 0.1-100 molar equivalents, preferably, 0.1-20molar equivalents, more preferably 0.1-5 molar equivalents, relative tocompound (1). The amount of compound (2) to be charged is 1-50 molarequivalents, preferably 1-10 molar equivalents, relative to compound(1).

The solvent to be used for the reaction of compound (1) and compound (2)is not particularly limited as long as it is a solvent or aqueoussolution that does not inhibit the reaction. For example, ethyl acetate,dichloromethane, chloroform, benzene, toluene and the like can bementioned. Among these, toluene and chloroform are preferable.

The reaction temperature is −20 to 200° C., preferably 0 to 80° C., morepreferably 20 to 50° C., and the reaction time is 1-48 hr, preferably2-24 hr.

When the reaction product of compound (1) and compound (2) is reactedwith compound (3), an alkali catalyst such as potassium carbonate,sodium carbonate, potassium t-butoxide and the like, or an acid catalystsuch as PTS (p-toluenesulfonic acid), MSA (methanesulfonic acid) and thelike may be used, like the catalyst used for the reaction of compound(1) and compound (2), or the reaction may be performed without acatalyst.

In addition, the reaction product of compound (1) and compound (2) maybe directly reacted with compound (3) by using a condensing agent suchas DCC (dicyclohexylcarbodiimide), DIC (diisopropylcarbodiimide), EDC(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) and thelike. Alternatively, compound (3) may be treated with a condensing agentto be once converted to an anhydride and the like, after which it isreacted with the reaction product of compound (1) and compound (2).

The amount of compound (3) to be charged is 1-50 molar equivalents,preferably 1-10 molar equivalents, relative to the reaction product ofcompound (1) and compound (2).

The catalyst to be used is appropriately selected according to thefunctional groups to be reacted.

The amount of catalyst is 0.05-100 molar equivalents, preferably 0.1-20molar equivalents, more preferably 0.2-5 molar equivalent, relative tocompound (1).

The solvent to be used for the reaction of the reaction product ofcompound (1) and compound (2) with compound (3) is not particularlylimited as long as it is a solvent or aqueous solution that does notinhibit the reaction. For example, ethyl acetate, dichloromethane,chloroform, benzene, toluene and the like can be mentioned. Among these,toluene and chloroform are preferable.

The reaction temperature is 0 to 200° C., preferably 0 to 120° C., morepreferably 20 to 50° C., and the reaction time is 1 hr-48 hr, preferably2-24 hr.

The reaction product obtained by the above-mentioned reaction can beappropriately purified by a general purification method, for example,washing with water, silica gel column chromatography, crystallization,recrystallization, liquid-liquid extraction, reprecipitation, ionexchange column chromatography and the like.

5.3 Structure XXIII Lipids

In some embodiments, an ionizable lipid is described in U.S. Pat. No.9,765,022.

The present invention provides a compound represented by structure(XXIII):

In structure XXIII, a hydrophilic and optionally positively charged headis

in which each of R_(a), R_(a)′, R_(a)″, and R_(a)′″, independently, isH, a C₁-C₂₀ monovalent aliphatic radical, a C₁-C₂₀ monovalentheteroaliphatic radical, a monovalent aryl radical, or a monovalentheteroaryl radical, and Z is a C₁-C₂₀ bivalent aliphatic radical, aC₁-C₂₀ bivalent heteroaliphatic radical, a bivalent aryl radical, or abivalent heteroaryl radical; B is a C₁-C₂₄ monovalent aliphatic radical,a C₁-C₂₄ monovalent heteroaliphatic radical, a monovalent aryl radical,a monovalent heteroaryl radical, or

each of R₁ and R₄, independently, is a bond, a C₁-C₁₀ bivalent aliphaticradical, a C₁-C₁₀ bivalent heteroaliphatic radical, a bivalent arylradical, or a bivalent heteroaryl radical; each of R₂ and R₅,independently, is a bond, a C₁-C₂₀ bivalent aliphatic radical, a C₁-C₂₀bivalent heteroaliphatic radical, a bivalent aryl radical, or a bivalentheteroaryl radical; each of R₃ and R₆, independently, is a C₁-C₂₀monovalent aliphatic radical, a C₁-C₂₀ monovalent heteroaliphaticradical, a monovalent aryl radical, or a monovalent heteroaryl radical;each of

a hydrophobic tail, and

also a hydrophobic tail, has 8 to 24 carbon atoms; and each of X alinker, and Y, also a linker, independently, is

in which each of m, n, p, q, and t, independently, is 1-6; W is O, S, orNR_(c); each of L₁, L₃, L₅, L₇, and L₉, directly linked to R₁, R₂, R₄,or R₅, independently, is a bond, O, S, or NR_(d); each of L₂, L₄, L₆,L₈, and L₁₀, independently, is a bond, O, S, or NR_(e); V is OR_(f),SR_(g), or NR_(h)R_(i); and each of R_(b), R_(c), R_(d), R_(e), R_(f),R_(g), R_(h), and R_(i), independently, is H, OH, C₁₋₁₀ oxyaliphaticradical, C₁-C₁₀ monovalent aliphatic radical, C₁-C₁₀ monovalentheteroaliphatic radical, a monovalent aryl radical, or a monovalentheteroaryl radical.

A subset of the above-described lipid-like compounds include those inwhich A is

each of R_(a) and R_(a)′, independently, being a C₁-C₁₀ monovalentaliphatic radical, a C₁-C₁₀ monovalent heteroaliphatic radical, amonovalent aryl radical, or a monovalent heteroaryl radical; and Z beinga C₁-C₁₀ bivalent aliphatic radical, a C₁-C₁₀ bivalent heteroaliphaticradical, a bivalent aryl radical, or a bivalent heteroaryl radical.

Some lipid-like compounds of this invention feature each of R₁ and R₄,independently, being C₁-C₆ (e.g., C₁-C₄) bivalent aliphatic radical or aC₁-C₆ (e.g., C₁-C₄) bivalent heteroaliphatic radical, the total carbonnumber for R₂ and R₃ being 12-20 (e.g., 14-18), the total carbon numberof R₅ and R₆ also being 12-20 (e.g., 14-18), and each of X and Y,independently, is

Specific examples of X and Y include

m being 2-6.

Still within the scope of this invention is a pharmaceutical compositioncontaining a nanocomplex that is formed of a protein and a bioreduciblecompound. In this pharmaceutical composition, the nanocomplex has aparticle size of 50 to 500 nm; the bioreducible compound contains adisulfide hydrophobic moiety, a hydrophilic moiety, and a linker joiningthe disulfide hydrophobic moiety and the hydrophilic moiety; and theprotein binds to the bioreducible compound via a non-covalentinteraction, a covalent bond, or both.

In certain embodiments, the disulfide hydrophobic moiety is aheteroaliphatic radical containing one or more —S—S— groups and 8 to 24carbon atoms; the hydrophilic moiety is an aliphatic or heteroaliphaticradical containing one or more hydrophilic groups and 1-20 carbon atoms,each of the hydrophilic groups being amino, alkylamino, dialkylamino,trialkylamino, tetraalkylammonium, hydroxyamino, hydroxyl, carboxyl,carboxylate, carbamate, carbamide, carbonate, phosphate, phosphite,sulfate, sulfite, or thiosulfate; and the linker is O, S, Si, C₁-C₆alkylene,

in which the variables are defined above.

Specific examples of X and Y include O, S, Si, C₁-C₆ alkylene,

In some embodiments, a lipid-like compound of this invention, as shownin structure XXIII above, includes (i) a hydrophilic head, A; (ii) ahydrophobic tail, R₂—S—S—R₃ and (iii) a linker, X. Optionally, thesecompounds contain a second hydrophobic tail, R₅—S—S—R₆ and a secondlinker, Y.

The hydrophilic head of structure XXIII contains one or more hydrophilicfunctional groups, e.g., hydroxyl, carbonyl, carboxyl, amino,sulfhydryl, phosphate, amide, ester, ether, carbamate, carbonate,carbamide, and phosphodiester. These groups can form hydrogen bonds andare optionally positively or negatively charged.

Examples of the hydrophilic head include:

Other examples include those described in Akinc et al., NatureBiotechnology, 26, 561-69 (2008) and Mahon et al., US Patent ApplicationPublication 2011/0293703.

The hydrophobic tail of structure XXIII is a saturated or unsaturated,linear or branched, acyclic or cyclic, aromatic or nonaromatichydrocarbon moiety containing a disulfide bond and 8-24 carbon atoms.One or more of the carbon atoms can be replaced with a heteroatom, suchas N, O, P, B, S, Si, Sb, Al, Sn, As, Se, and Ge. The tail is optionallysubstituted with one or more groups described above. The lipid-likecompounds containing this disulfide bond can be bioreducible.

Examples include:

A linker of structure XXIII links the hydrophilic head and thehydrophobic tail. The linker can be any chemical group that ishydrophilic or hydrophobic, polar or non-polar, e.g., 0, S, Si, amino,alkylene, ester, amide, carbamate, carbamide, carbonate, phosphate,phosphite, sulfate, sulfite, and thiosulfate. Examples include:

Shown below are exemplary lipid-like compounds of this invention:

The lipid-like compounds of structure XXIII can be prepared by methodswell known the art. See Wang et al., ACS Synthetic Biology, 1, 403-07(2012); Manoharan, et al., International Patent Application PublicationWO 2008/042973; and Zugates et al., U.S. Pat. No. 8,071,082. The routeshown below exemplifies synthesis of these lipid-like compounds:

Each of L_(a), L_(a)′, L, and L′ can be one of L₁-L₁₀; each of W_(a) andW_(b), independently, is W or V; and R_(a) and R₁-R₆ are defined above,as well as L₁-L₁₀, W, and V.

In this exemplary synthetic route, an amine compound, i.e., compound D,reacts with bromides E1 and E2 to form compound F, which is then coupledwith both G1 and G2 to afford the final product, i.e., compound H. Oneor both of the double bonds in this compound (shown above) can bereduced to one or two single bonds to obtain different lipid-likecompounds of structure XXIII.

Other lipid-like compounds of this invention can be prepared using othersuitable starting materials through the above-described synthetic routeand others known in the art. The method set forth above can include anadditional step(s) to add or remove suitable protecting groups in orderto ultimately allow synthesis of the lipid-like compounds. In addition,various synthetic steps can be performed in an alternate sequence ororder to give the desired material. Synthetic chemistry transformationsand protecting group methodologies (protection and deprotection) usefulin synthesizing applicable lipid-like compounds are known in the art,including, for example, R. Larock, Comprehensive Organic Transformations(2nd Ed., VCH Publishers 1999); P. G. M. Wuts and T. W. Greene, Greene'sProtective Groups in Organic Synthesis (4th Ed., John Wiley and Sons2007); L. Fieser and M Fieser, Fieser and Fieser's Reagents for OrganicSynthesis (John Wiley and Sons 1994); and L. Paquette, ed., Encyclopediaof Reagents for Organic Synthesis (2nd ed., John Wiley and Sons 2009)and subsequent editions thereof. Certain lipid-like compounds maycontain a non-aromatic double bond and one or more asymmetric centers.Thus, they can occur as non-aromatic double bond and one or moreasymmetric centers. Thus, they can occur as racemates and racemicmixtures, single enantiomers, individual diastereomers, diastereomericmixtures, and cis- or trans-isomeric forms. All such isomeric forms arecontemplated.

As mentioned above, these lipid-like compounds are useful for deliveryof pharmaceutical agents. They can be preliminarily screened for theirefficacy in delivering pharmaceutical agents by an in vitro assay andthen confirmed by animal experiments and clinic trials. Other methodswill also be apparent to those of ordinary skill in the art.

Not to be bound by any theory, the lipid-like compounds of structureXXIII facilitate delivery of pharmaceutical agents by forming complexes,e.g., nanocomplexes and microparticles. The hydrophilic head of such alipid-like compound, positively or negatively charged, binds to a moietyof a pharmaceutical agent that is oppositely charged and its hydrophobicmoiety binds to a hydrophobic moiety of the pharmaceutical agent. Eitherbinding can be covalent or non-covalent.

The above described complexes can be prepared using procedures describedin publications such as Wang et al., ACS Synthetic Biology, 1, 403-07(2012). Generally, they are obtained by incubating a lipid-like compoundand a pharmaceutical agent in a buffer such as a sodium acetate bufferor a phosphate buffered saline (“PBS”).

5.4 Hydrophilic Groups

In certain embodiments, the selected hydrophilic functional group ormoiety may alter or otherwise impart properties to the compound or tothe transfer vehicle of which such compound is a component (e.g., byimproving the transfection efficiencies of a lipid nanoparticle of whichthe compound is a component). For example, the incorporation ofguanidinium as a hydrophilic head-group in the compounds disclosedherein may promote the fusogenicity of such compounds (or of thetransfer vehicle of which such compounds are a component) with the cellmembrane of one or more target cells, thereby enhancing, for example,the transfection efficiencies of such compounds. It has beenhypothesized that the nitrogen from the hydrophilic guanidinium moietyforms a six-membered ring transition state which grants stability to theinteraction and thus allows for cellular uptake of encapsulatedmaterials. (Wender, et al., Adv. Drug Del. Rev. (2008) 60: 452-472.)Similarly, the incorporation of one or more amino groups or moietiesinto the disclosed compounds (e.g., as a head-group) may further promotedisruption of the endosomal/lysosomal membrane of the target cell byexploiting the fusogenicity of such amino groups. This is based not onlyon the pKa of the amino group of the composition, but also on theability of the amino group to undergo a hexagonal phase transition andfuse with the target cell surface, i.e. the vesicle membrane. (Koltover,et al. Science (1998) 281: 78-81.) The result is believed to promote thedisruption of the vesicle membrane and release of the lipid nanoparticlecontents into the target cell.

Similarly, in certain embodiments the incorporation of, for example,imidazole as a hydrophilic head-group in the compounds disclosed hereinmay serve to promote endosomal or lysosomal release of, for example,contents that are encapsulated in a transfer vehicle (e.g., lipidnanoparticle) of the invention. Such enhanced release may be achieved byone or both of a proton-sponge mediated disruption mechanism and/or anenhanced fusogenicity mechanism. The proton-sponge mechanism is based onthe ability of a compound, and in particular a functional moiety orgroup of the compound, to buffer the acidification of the endosome. Thismay be manipulated or otherwise controlled by the pKa of the compound orof one or more of the functional groups comprising such compound (e.g.,imidazole). Accordingly, in certain embodiments the fusogenicity of, forexample, the imidazole-based compounds disclosed herein (e.g., HGT4001and HGT4004) are related to the endosomal disruption properties, whichare facilitated by such imidazole groups, which have a lower pKarelative to other traditional ionizable lipids. Such endosomaldisruption properties in turn promote osmotic swelling and thedisruption of the liposomal membrane, followed by the transfection orintracellular release of the polynucleotide materials loaded orencapsulated therein into the target cell. This phenomenon can beapplicable to a variety of compounds with desirable pKa profiles inaddition to an imidazole moiety. Such embodiments also includemulti-nitrogen based functionalities such as polyamines, polypeptide(histidine), and nitrogen-based dendritic structures.

Exemplary ionizable and/or cationic lipids are described inInternational PCT patent publications WO2015/095340, WO2015/199952,WO2018/011633, WO2017/049245, WO2015/061467, WO2012/040184,WO2012/000104, WO2015/074085, WO2016/081029, WO2017/004143,WO2017/075531, WO2017/117528, WO2011/022460, WO2013/148541,WO2013/116126, WO2011/153120, WO2012/044638, WO2012/054365,WO2011/090965, WO2013/016058, WO2012/162210, WO2008/042973,WO2010/129709, WO2010/144740, WO2012/099755, WO2013/049328,WO2013/086322, WO2013/086373, WO2011/071860, WO2009/132131,WO2010/048536, WO2010/088537, WO2010/054401, WO2010/054406,WO2010/054405, WO2010/054384, WO2012/016184, WO2009/086558,WO2010/042877, WO2011/000106, WO2011/000107, WO2005/120152,WO2011/141705, WO2013/126803, WO2006/007712, WO2011/038160,WO2005/121348, WO2011/066651, WO2009/127060, WO2011/141704,WO2006/069782, WO2012/031043, WO2013/006825, WO2013/033563,WO2013/089151, WO2017/099823, WO2015/095346, and WO2013/086354, and USpatent publications US2016/0311759, US2015/0376115, US2016/0151284,US2017/0210697, US2015/0140070, US2013/0178541, US2013/0303587,US2015/0141678, US2015/0239926, US2016/0376224, US2017/0119904,US2012/0149894, US2015/0057373, US2013/0090372, US2013/0274523,US2013/0274504, US2013/0274504, US2009/0023673, US2012/0128760,US2010/0324120, US2014/0200257, US2015/0203446, US2018/0005363,US2014/0308304, US2013/0338210, US2012/0101148, US2012/0027796,US2012/0058144, US2013/0323269, US2011/0117125, US2011/0256175,US2012/0202871, US2011/0076335, US2006/0083780, US2013/0123338,US2015/0064242, US2006/0051405, US2013/0065939, US2006/0008910,US2003/0022649, US2010/0130588, US2013/0116307, US2010/0062967,US2013/0202684, US2014/0141070, US2014/0255472, US2014/0039032,US2018/0028664, US2016/0317458, and US2013/0195920, the contents of allof which are incorporated herein by reference in their entirety.International patent application WO 2019/131770 is also incorporatedherein by reference in its entirety.

6. PEG Lipids

The use and inclusion of polyethylene glycol (PEG)-modifiedphospholipids and derivatized lipids such as derivatized ceramides(PEG-CER), including N-Octanoyl-Sphingosine-1-[Succinyl(MethoxyPolyethylene Glycol)-2000] (C8 PEG-2000 ceramide) in the liposomal andpharmaceutical compositions described herein is contemplated, preferablyin combination with one or more of the compounds and lipids disclosedherein. Contemplated PEG-modified lipids include, but are not limitedto, a polyethylene glycol chain of up to 5 kDa in length covalentlyattached to a lipid with alkyl chain(s) of C6-C20 length. In someembodiments, the PEG-modified lipid employed in the compositions andmethods of the invention is 1,2-dimyristoyl-sn-glycerol,methoxypolyethylene Glycol (2000 MW PEG) “DMG-PEG2000.” The addition ofPEG-modified lipids to the lipid delivery vehicle may prevent complexaggregation and may also provide a means for increasing circulationlifetime and increasing the delivery of the lipid-polynucleotidecomposition to the target tissues, (Klibanov et al. (1990) FEBS Letters,268 (1): 235-237), or they may be selected to rapidly exchange out ofthe formulation in vivo (see U.S. Pat. No. 5,885,613). Particularlyuseful exchangeable lipids are PEG-ceramides having shorter acyl chains(e.g., C14 or C18). The PEG-modified phospholipid and derivatized lipidsof the present invention may comprise a molar ratio from about 0% toabout 20%, about 0.5% to about 20%, about 1% to about 15%, about 4% toabout 10%, or about 2% of the total lipid present in a liposomal lipidnanoparticle.

In an embodiment, a PEG-modified lipid is described in InternationalPat. Appl. No. PCT/US2019/015913, which is incorporated herein byreference in their entirety. In an embodiment, a transfer vehiclecomprises one or more PEG-modified lipids.

Non-limiting examples of PEG-modified lipids include PEG-modifiedphosphatidylethanolamines and phosphatidic acids, PEG-ceramideconjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylaminesand PEG-modified 1,2-diacyloxypropan-3-amines. In some furtherembodiments, a PEG-modified lipid may be, e.g., PEG-c-DOMG, PEG-DMG,PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE.

In some still further embodiments, the PEG-modified lipid includes, butis not limited to 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol(PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disterylglycerol (PEG-DSG),PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide(PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), orPEG-1,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA).

In various embodiments, a PEG-modified lipid may also be referred to as“PEGylated lipid” or “PEG-lipid.”

In one embodiment, the PEG-lipid is selected from the group consistingof a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidicacid, a PEG-modified ceramide, a PEG-modified dialkylamine, aPEG-modified diacylglycerol, a PEG-modified dialkylglycerol, andmixtures thereof.

In some embodiments, the lipid moiety of the PEG-lipids includes thosehaving lengths of from about C₁₄ to about C₂₂, such as from about C₁₄ toabout C₁₆. In some embodiments, a PEG moiety, for example a mPEG-NH₂,has a size of about 1000, about 2000, about 5000, about 10,000, about15,000 or about 20,000 daltons. In one embodiment, the PEG-lipid isPEG2k-DMG.

In one embodiment, the lipid nanoparticles described herein can comprisea lipid modified with a non-diffusible PEG. Non-limiting examples ofnon-diffusible PEGs include PEG-DSG and PEG-DSPE.

PEG-lipids are known in the art, such as those described in U.S. Pat.No. 8,158,601 and International Pat. Publ. No. WO2015/130584 A2, whichare incorporated herein by reference in their entirety.

In various embodiments, lipids (e.g., PEG-lipids), described herein maybe synthesized as described International Pal. Publ. No.PCT/US2016/000129, which is incorporated by reference in its entirety.

The lipid component of a lipid nanoparticle composition may include oneor more molecules comprising polyethylene glycol, such as PEG orPEG-modified lipids. Such species may be alternately referred to asPEGylated lipids. A PEG lipid is a lipid modified with polyethyleneglycol. A PEG lipid may be selected from the non-limiting groupincluding PEG-modified phosphatidylethanolamines, PEG-modifiedphosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines,PEG-modified diacylglycerols, PEG-modified dialkylglycerols, andmixtures thereof. For example, a PEG lipid may be PEG-c-DOMG, PEG-DMG,PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.

In some embodiments the PEG-modified lipids are a modified form ofPEG-DMG. PEG-DMG has the following structure:

In some embodiments the PEG-modified lipids are a modified form ofPEG-C18, or PEG-1. PEG-1 has the following structure

In one embodiment, PEG lipids useful in the present invention can bePEGylated lipids described in International Publication No.WO2012099755, the contents of which is herein incorporated by referencein its entirety. Any of these exemplary PEG lipids described herein maybe modified to comprise a hydroxyl group on the PEG chain. In certainembodiments, the PEG lipid is a PEG-OH lipid. In certain embodiments,the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain.In certain embodiments, a PEG-OH or hydroxy-PEGylated lipid comprises an—OH group at the terminus of the PEG chain. Each possibility representsa separate embodiment of the present invention.

In some embodiments, the PEG lipid is a compound of Formula (P1):

or a salt or isomer thereof, wherein:

-   -   r is an integer between 1 and 100;    -   R is C₁₀₋₄₀ alkyl, C₁₀₋₄₀ alkenyl, or C₁₀₋₄₀ alkynyl; and        optionally one or more methylene groups of R are independently        replaced with C₃₋₁₀ carbocyclylene, 4 to 10 membered        heterocyclylene, C₆₋₁₀ arylene, 4 to 10 membered heteroarylene,        —N(R^(N))—, —O—, —S—, —C(O)—, —C(O)N(R^(N))—, —NR^(N)C(O)—,        —NR^(N)C(O)N(R^(N))—, —C(O)O—, —OC(O)—, —OC(O)O—,        OC(O)N(R^(N))—, —NR^(N)C(O)O—, —C(O)S—, —SC(O)—, —C(═NR^(N))—,        —C(═NR^(N))N(R^(N))—, —NR^(N)C(═NR^(N))—,        —NR^(N)C(═NR^(N))N(R^(N))—, —C(S)—, —C(S)N(R^(N))—,        —NR^(N)C(S)—, —NR^(N)C(S)N(R^(N))—, —S(O)—, —OS(O)—, —S(O)O—,        —OS(O)O—, —OS(O)₂—, —S(O)₂O—, —OS(O)₂O—, —N(R^(N))S(O)—,        —S(O)N(R^(N))—, —N(R^(N))S(O)N(R^(N))—, —OS(O)N(R^(N))—,        —N(R^(N))S(O)O—, —S(O)₂—, —N(R^(N))S(O)₂—, —S(O)₂N(R^(N))—,        —N(R^(N))S(O)₂N(R^(N))—, —OS(O)₂N(R^(N))—, or —N(R^(N))S(O)₂O—;        and    -   each instance of R^(N) is independently hydrogen, C₁₋₆ alkyl, or        a nitrogen protecting group.

For example, R is C17 alkyl. For example, the PEG lipid is a compound ofFormula (P1-a):

or a salt or isomer thereof, wherein r is an integer between 1 and 100.

For example, the PEG lipid is a compound of the following formula:

1. Helper Lipids

In some embodiments, the transfer vehicle (e.g., LNP) described hereincomprises one or more non-cationic helper lipids. In some embodiments,the helper lipid is a phospholipid. In some embodiments, the helperlipid is a phospholipid substitute or replacement. In some embodiments,the phospholipid or phospholipid substitute can be, for example, one ormore saturated or (poly)unsaturated phospholipids, or phospholipidsubstitutes, or a combination thereof. In general, phospholipidscomprise a phospholipid moiety and one or more fatty acid moieties.

A phospholipid moiety can be selected, for example, from thenon-limiting group consisting of phosphatidyl choline, phosphatidylethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidicacid, 2-lysophosphatidyl choline, and a sphingomyelin.

A fatty acid moiety can be selected, for example, from the non-limitinggroup consisting of lauric acid, myristic acid, myristoleic acid,palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleicacid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid,arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoicacid, and docosahexaenoic acid.

Phospholipids include, but are not limited to, glycerophospholipids suchas phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines,phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids.Phospholipids also include phosphosphingolipid, such as sphingomyelin.

In some embodiments, the helper lipid is a1,2-distearoyl-177-glycero-3-phosphocholine (DSPC) analog, a DSPCsubstitute, oleic acid, or an oleic acid analog.

In some embodiments, a helper lipid is a non-phosphatidyl choline (PC)zwitterionic lipid, a DSPC analog, oleic acid, an oleic acid analog, ora DSPC substitute.

In some embodiments, a helper lipid is described in PCT/US2018/053569.Helper lipids suitable for use in a lipid composition of the disclosureinclude, for example, a variety of neutral, uncharged or zwitterioniclipids. Such helper lipids are preferably used in combination with oneor more of the compounds and lipids disclosed herein. Examples of helperlipids include, but are not limited to, 5-heptadecylbenzene-1,3-diol(resorcinol), dipalmitoylphosphatidylcholine (DPPC),distearoylphosphatidylcholine (DSPC), pohsphocholine (DOPC),dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC),1,2-distearoylsn-glycero-3-phosphocholine (DAPC),phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC),dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine(DMPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (MPPC),1-paimitoyl-2-myristoyl phosphatidylcholine (PMPC),1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC),1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC),1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC),1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), paimitoyioieoylphosphatidylcholine (POPC), lysophosphatidyl choline, dioleoylphosphatidylethanol amine (DOPE) dilinoleoylphosphatidylcholinedistearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine(DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE),lysophosphatidylethanolamine and combinations thereof. In oneembodiment, the helper lipid may be distearoylphosphatidylcholine (DSPC)or dimyristoyl phosphatidyl ethanolamine (DMPE). In another embodiment,the helper lipid may be distearoylphosphatidylcholine (DSPC). Helperlipids function to stabilize and improve processing of the transfervehicles. Such helper lipids are preferably used in combination withother excipients, for example, one or more of the ionizable lipidsdisclosed herein. In some embodiments, when used in combination with anionizable lipid, the helper lipid may comprise a molar ratio of 5% toabout 90%, or about 10% to about 70% of the total lipid present in thelipid nanoparticle.

8. Structural Lipids

In an embodiment, a structural lipid is described in internationalpatent application PCT/US2019/015913.

The transfer vehicles described herein comprise one or more structurallipids. Incorporation of structural lipids in the lipid nanoparticle mayhelp mitigate aggregation of other lipids in the particle. Structurallipids can include, but are not limited to, cholesterol, fecosterol,ergosterol, bassicasterol, tomatidine, tomatine, ursolic,alpha-tocopherol, and mixtures thereof. In certain embodiments, thestructural lipid is cholesterol. In certain embodiments, the structurallipid includes cholesterol and a corticosteroid (such as, for example,prednisolone, dexamethasone, prednisone, and hydrocortisone), or acombination thereof.

In some embodiments, the structural lipid is a sterol. In certainembodiments, the structural lipid is a steroid. In certain embodiments,the structural lipid is cholesterol. In certain embodiments, thestructural lipid is an analog of cholesterol. In certain embodiments,the structural lipid is alpha-tocopherol.

The transfer vehicles described herein comprise one or more structurallipids. Incorporation of structural lipids in a transfer vehicle, e.g.,a lipid nanoparticle, may help mitigate aggregation of other lipids inthe particle. In certain embodiments, the structural lipid includescholesterol and a corticosteroid (such as, for example, prednisolone,dexamethasone, prednisone, and hydrocortisone), or a combinationthereof.

In some embodiments, the structural lipid is a sterol. Structural lipidscan include, but are not limited to, sterols (e.g., phytosterols orzoosterols).

In certain embodiments, the structural lipid is a steroid. For example,sterols can include, but are not limited to, cholesterol, β-sitosterol,fecosterol, ergosterol, sitosterol, campesterol, stigmasterol,brassicasterol, ergosterol, tomatidine, tomatine, ursolic acid, oralpha-tocopherol.

In some embodiments, a transfer vehicle includes an effective amount ofan immune cell delivery potentiating lipid, e.g., a cholesterol analogor an amino lipid or combination thereof, that, when present in atransfer vehicle, e.g., an lipid nanoparticle, may function by enhancingcellular association and/or uptake, internalization, intracellulartrafficking and/or processing, and/or endosomal escape and/or mayenhance recognition by and/or binding to immune cells, relative to atransfer vehicle lacking the immune cell delivery potentiating lipid.Accordingly, while not intending to be bound by any particular mechanismor theory, in one embodiment, a structural lipid or other immune celldelivery potentiating lipid of the disclosure binds to C1q or promotesthe binding of a transfer vehicle comprising such lipid to C1q. Thus,for in vitro use of the transfer vehicles of the disclosure for deliveryof a nucleic acid molecule to an immune cell, culture conditions thatinclude C1q are used (e.g., use of culture media that includes serum oraddition of exogenous C1q to serum-free media). For in vivo use of thetransfer vehicles of the disclosure, the requirement for C1q is suppliedby endogenous C1q.

In certain embodiments, the structural lipid is cholesterol. In certainembodiments, the structural lipid is an analog of cholesterol. In someembodiments, the structural lipid is a lipid in Table 16:

TABLE 16 CMPD No. S- Structure 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

150

154

162

163

164

184

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

165

169

170

171

172

43

44

45

46

175

176

51

52

53

54

55

56

57

58

59

153

47

48

49

50

177

178

60

61

62

63

64

65

66

67

149

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

87

152

157

88

89

90

91

93

94

95

96

92

98

99

100

101

97

102

103

104

105

180

181

182

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

156

158

160

161

166

121

122

123

124

125

126

127

128

129

130

155

167

168

173

174

179

131

132

133

134

135

136

137

138

139

140

141

159

142

143

144

145

146

147

148

151

183

9. LNP Formulations

The formation of a lipid nanoparticle (LNP) described herein may beaccomplished by any methods known in the art. For example, as describedin U.S. Pat. Pub. No. US2012/0178702 A1, which is incorporated herein byreference in its entirety. Non-limiting examples of lipid nanoparticlecompositions and methods of making them are described, for example, inSemple et al. (2010) Nat. Biotechnol. 28:172-176; Jayarama et al.(2012), Angew. Chem. Int. Ed., 51:8529-8533; and Maier et al. (2013)Molecular Therapy 21, 1570-1578 (the contents of each of which areincorporated herein by reference in their entirety).

In one embodiment, the LNP formulation may be prepared by, e.g., themethods described in International Pat. Pub. No. WO 2011/127255 or WO2008/103276, the contents of each of which are herein incorporated byreference in their entirety.

In one embodiment, LNP formulations described herein may comprise apolycationic composition. As a non-limiting example, the polycationiccomposition may be a composition selected from Formulae 1-60 of U.S.Pat. Pub. No. US2005/0222064 A1, the content of which is hereinincorporated by reference in its entirety.

In one embodiment, the lipid nanoparticle may be formulated by themethods described in U.S. Pat. Pub. No. US2013/0156845 A1, andInternational Pat. Pub. No. WO2013/093648 A2 or WO2012/024526 A2, eachof which is herein incorporated by reference in its entirety.

In one embodiment, the lipid nanoparticles described herein may be madein a sterile environment by the system and/or methods described in U.S.Pat. Pub. No. US2013/0164400 A1, which is incorporated herein byreference in its entirety.

In one embodiment, the LNP formulation may be formulated in ananoparticle such as a nucleic acid-lipid particle described in U.S.Pat. No. 8,492,359, which is incorporated herein by reference in itsentirety.

A nanoparticle composition may optionally comprise one or more coatings.For example, a nanoparticle composition may be formulated in a capsule,film, or tablet having a coating. A capsule, film, or tablet including acomposition described herein may have any useful size, tensile strength,hardness, or density.

In some embodiments, the lipid nanoparticles described herein may besynthesized using methods comprising microfluidic mixers. Exemplarymicrofluidic mixers may include, but are not limited to, a slitinterdigitial micromixer including, but not limited to, thosemanufactured by Precision Nanosystems (Vancouver, BC, Canada),Microinnova (Allerheiligen bei Wildon, Austria) and/or a staggeredherringbone micromixer (SHM) (Zhigaltsev, I. V. et al. (2012) Langmuir.28:3633-40; Belliveau, N. M. et al. Mol. Ther. Nucleic. Acids. (2012)1:e37; Chen, D. et al. J. Am. Chem. Soc. (2012) 134(16):6948-51; each ofwhich is herein incorporated by reference in its entirety).

In some embodiments, methods of LNP generation comprising SHM, furthercomprise the mixing of at least two input streams wherein mixing occursby microstructure-induced chaotic advection (MICA). According to thismethod, fluid streams flow through channels present in a herringbonepattern causing rotational flow and folding the fluids around eachother. This method may also comprise a surface for fluid mixing whereinthe surface changes orientations during fluid cycling. Methods ofgenerating LNPs using SHM include those disclosed in U.S. Pat. Pub. Nos.US2004/0262223 A1 and US2012/0276209 A1, each of which is incorporatedherein by reference in their entirety.

In one embodiment, the lipid nanoparticles may be formulated using amicromixer such as, but not limited to, a Slit InterdigitalMicrostructured Mixer (SIMM-V2) or a Standard Slit Interdigital MicroMixer (SSIMM) or Caterpillar (CPMM) or Impinging-jet (IJMM) from theInstitut fur Mikrotechnik Mainz GmbH, Mainz Germany). In one embodiment,the lipid nanoparticles are created using microfluidic technology (see,Whitesides (2006) Nature. 442: 368-373; and Abraham et al. (2002)Science. 295: 647-651; each of which is herein incorporated by referencein its entirety). As a non-limiting example, controlled microfluidicformulation includes a passive method for mixing streams of steadypressure-driven flows in micro channels at a low Reynolds number (see,e.g., Abraham et al. (2002) Science. 295: 647651; which is hereinincorporated by reference in its entirety).

In one embodiment, the circRNA of the present invention may beformulated in lipid nanoparticles created using a micromixer chip suchas, but not limited to, those from Harvard Apparatus (Holliston, Mass.),Dolomite Microfluidics (Royston, UK), or Precision Nanosystems (VanCouver, BC, Canada). A micromixer chip can be used for rapid mixing oftwo or more fluid streams with a split and recombine mechanism.

In one embodiment, the lipid nanoparticles may have a diameter fromabout 10 to about 100 nm such as, but not limited to, about 10 to about20 nm, about 10 to about 30 nm, about 10 to about 40 nm, about 10 toabout 50 nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10to about 80 nm, about 10 to about 90 nm, about 20 to about 30 nm, about20 to about 40 nm, about 20 to about 50 nm, about 20 to about 60 nm,about 20 to about 70 nm, about 20 to about 80 nm, about 20 to about 90nm, about 20 to about 100 nm, about 30 to about 40 nm, about 30 to about50 nm, about 30 to about 60 nm, about 30 to about 70 nm, about 30 toabout 80 nm, about 30 to about 90 nm, about 30 to about 100 nm, about 40to about 50 nm, about 40 to about 60 nm, about 40 to about 70 nm, about40 to about 80 nm, about 40 to about 90 nm, about 40 to about 100 nm,about 50 to about 60 nm, about 50 to about 70 nm about 50 to about 80nm, about 50 to about 90 nm, about 50 to about 100 nm, about 60 to about70 nm, about 60 to about 80 nm, about 60 to about 90 nm, about 60 toabout 100 nm, about 70 to about 80 nm, about 70 to about 90 nm, about 70to about 100 nm, about 80 to about 90 nm, about 80 to about 100 nmand/or about 90 to about 100 nm. In one embodiment, the lipidnanoparticles may have a diameter from about 10 to 500 nm. In oneembodiment, the lipid nanoparticle may have a diameter greater than 100nm, greater than 150 nm, greater than 200 nm, greater than 250 nm,greater than 300 nm, greater than 350 nm, greater than 400 nm, greaterthan 450 nm, greater than 500 nm, greater than 550 nm, greater than 600nm, greater than 650 nm, greater than 700 nm, greater than 750 nm,greater than 800 nm, greater than 850 nm, greater than 900 nm, greaterthan 950 nm or greater than 1000 nm. Each possibility represents aseparate embodiment of the present invention.

In some embodiments, a nanoparticle (e.g., a lipid nanoparticle) has amean diameter of 10-500 nm, 20-400 nm, 30-300 nm, or 40-200 nm. In someembodiments, a nanoparticle (e.g., a lipid nanoparticle) has a meandiameter of 50-150 nm, 50-200 nm, 80-100 nm, or 80-200 nm.

In some embodiments, the lipid nanoparticles described herein can have adiameter from below 0.1 μm to up to 1 mm such as, but not limited to,less than 0.1 μm, less than 1.0 μm, less than 5 μm, less than 10 μm,less than 15 μm, less than 20 μm, less than 25 μm, less than 30 μm, lessthan 35 μm, less than 40 μm, less than 50 μm, less than 55 μm, less than60 μm, less than 65 μm, less than 70 μm, less than 75 μm, less than 80μm, less than 85 μm, less than 90 μm, less than 95 μm, less than 100 μm,less than 125 μm, less than 150 μm, less than 175 μm, less than 200 μm,less than 225 μm, less than 250 μm, less than 275 μm, less than 300 μm,less than 325 μm, less than 350 μm, less than 375 μm, less than 400 μm,less than 425 μm, less than 450 μm, less than 475 μm, less than 500 μm,less than 525 μm, less than 550 μm, less than 575 μm, less than 600 μm,less than 625 μm, less than 650 μm, less than 675 μm, less than 700 μm,less than 725 μm, less than 750 μm, less than 775 μm, less than 800 μm,less than 825 μm, less than 850 μm, less than 875 μm, less than 900 μm,less than 925 μm, less than 950 μm, less than 975 μm.

In another embodiment, LNPs may have a diameter from about 1 nm to about100 nm, from about 1 nm to about 10 nm, about 1 nm to about 20 nm, fromabout 1 nm to about 30 nm, from about 1 nm to about 40 nm, from about 1nm to about 50 nm, from about 1 nm to about 60 nm, from about 1 nm toabout 70 nm, from about 1 nm to about 80 nm, from about 1 nm to about 90nm, from about 5 nm to about from 100 nm, from about 5 nm to about 10nm, about 5 nm to about 20 nm, from about 5 nm to about 30 nm, fromabout 5 nm to about 40 nm, from about 5 nm to about 50 nm, from about 5nm to about 60 nm, from about 5 nm to about 70 nm, from about 5 nm toabout 80 nm, from about 5 nm to about 90 nm, about 10 to about 50 nM,from about 20 to about 50 nm, from about 30 to about 50 nm, from about40 to about 50 nm, from about 20 to about 60 nm, from about 30 to about60 nm, from about 40 to about 60 nm, from about 20 to about 70 nm, fromabout 30 to about 70 nm, from about 40 to about 70 nm, from about 50 toabout 70 nm, from about 60 to about 70 nm, from about 20 to about 80 nm,from about 30 to about 80 nm, from about 40 to about 80 nm, from about50 to about 80 nm, from about 60 to about 80 nm, from about 20 to about90 nm, from about 30 to about 90 nm, from about 40 to about 90 nm, fromabout 50 to about 90 nm, from about 60 to about 90 nm and/or from about70 to about 90 nm. Each possibility represents a separate embodiment ofthe present invention.

A nanoparticle composition may be relatively homogenous. Apolydispersity index may be used to indicate the homogeneity of ananoparticle composition, e.g., the particle size distribution of thenanoparticle compositions. A small (e.g., less than 0.3) polydispersityindex generally indicates a narrow particle size distribution. Ananoparticle composition may have a polydispersity index from about 0 toabout 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08,0.09, 0.10, 0.1 1, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20,0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersityindex of a nanoparticle composition may be from about 0.10 to about0.20. Each possibility represents a separate embodiment of the presentinvention.

The zeta potential of a nanoparticle composition may be used to indicatethe electrokinetic potential of the composition. For example, the zetapotential may describe the surface charge of a nanoparticle composition.Nanoparticle compositions with relatively low charges, positive ornegative, are generally desirable, as more highly charged species mayinteract undesirably with cells, tissues, and other elements in thebody. In some embodiments, the zeta potential of a nanoparticlecomposition may be from about −20 mV to about +20 mV, from about −20 mVto about +15 mV, from about −20 mV to about +10 mV, from about −20 mV toabout +5 mV, from about −20 mV to about 0 mV, from about −20 mV to about−5 mV, from about −20 mV to about −10 mV, from about −20 mV to about −15mV from about −20 mV to about +20 mV, from about −20 mV to about +15 mV,from about −20 mV to about +10 mV, from about −20 mV to about +5 mV,from about −20 mV to about 0 mV, from about 0 mV to about +20 mV, fromabout 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mVto about +15 mV, or from about +5 mV to about +10 mV. Each possibilityrepresents a separate embodiment of the present invention.

The efficiency of encapsulation of a therapeutic agent describes theamount of therapeutic agent that is encapsulated or otherwise associatedwith a nanoparticle composition after preparation, relative to theinitial amount provided. The encapsulation efficiency is desirably high(e.g., close to 100%). The encapsulation efficiency may be measured, forexample, by comparing the amount of therapeutic agent in a solutioncontaining the nanoparticle composition before and after breaking up thenanoparticle composition with one or more organic solvents ordetergents. Fluorescence may be used to measure the amount of freetherapeutic agent (e.g., nucleic acids) in a solution. For thenanoparticle compositions described herein, the encapsulation efficiencyof a therapeutic agent may be at least 50%, for example 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%. In some embodiments, the encapsulation efficiency may beat least 80%. In certain embodiments, the encapsulation efficiency maybe at least 90%. Each possibility represents a separate embodiment ofthe present invention. In some embodiments, the lipid nanoparticle has apolydiversity value of less than 0.4. In some embodiments, the lipidnanoparticle has a net neutral charge at a neutral pH. In someembodiments, the lipid nanoparticle has a mean diameter of 50-200 nm.

The properties of a lipid nanoparticle formulation may be influenced byfactors including, but not limited to, the selection of the cationiclipid component, the degree of cationic lipid saturation, the selectionof the non-cationic lipid component, the degree of noncationic lipidsaturation, the selection of the structural lipid component, the natureof the PEGylation, ratio of all components and biophysical parameterssuch as size. As described herein, the purity of a PEG lipid componentis also important to an LNP's properties and performance.

10. Methods

In one embodiment, a lipid nanoparticle formulation may be prepared bythe methods described in International Publication Nos. WO2011127255 orWO2008103276, each of which is herein incorporated by reference in theirentirety. In some embodiments, lipid nanoparticle formulations may be asdescribed in International Publication No. WO2019131770, which is hereinincorporated by reference in its entirety.

In some embodiments, circular RNA is formulated according to a processdescribed in U.S. patent application Ser. No. 15/809,680. In someembodiments, the present invention provides a process of encapsulatingcircular RNA in transfer vehicles comprising the steps of forming lipidsinto pre-formed transfer vehicles (i.e. formed in the absence of RNA)and then combining the pre-formed transfer vehicles with RNA. In someembodiments, the novel formulation process results in an RNA formulationwith higher potency (peptide or protein expression) and higher efficacy(improvement of a biologically relevant endpoint) both in vitro and invivo with potentially better tolerability as compared to the same RNAformulation prepared without the step of preforming the lipidnanoparticles (e.g., combining the lipids directly with the RNA).

For certain cationic lipid nanoparticle formulations of RNA, in order toachieve high encapsulation of RNA, the RNA in buffer (e.g., citratebuffer) has to be heated. In those processes or methods, the heating isrequired to occur before the formulation process (i.e. heating theseparate components) as heating post-formulation (post-formation ofnanoparticles) does not increase the encapsulation efficiency of the RNAin the lipid nanoparticles. In contrast, in some embodiments of thenovel processes of the present invention, the order of heating of RNAdoes not appear to affect the RNA encapsulation percentage. In someembodiments, no heating (i.e. maintaining at ambient temperature) of oneor more of the solutions comprising the pre-formed lipid nanoparticles,the solution comprising the RNA and the mixed solution comprising thelipid nanoparticle encapsulated RNA is required to occur before or afterthe formulation process.

RNA may be provided in a solution to be mixed with a lipid solution suchthat the RNA may be encapsulated in lipid nanoparticles. A suitable RNAsolution may be any aqueous solution containing RNA to be encapsulatedat various concentrations. For example, a suitable RNA solution maycontain an RNA at a concentration of or greater than about 0.01 mg/ml,0.05 mg/ml, 0.06 mg/ml, 0.07 mg/ml, 0.08 mg/ml, 0.09 mg/ml, 0.1 mg/ml,0.15 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7mg/ml, 0.8 mg/ml, 0.9 mg/ml, or 1.0 mg/ml. In some embodiments, asuitable RNA solution may contain an RNA at a concentration in a rangefrom about 0.01-1.0 mg/ml, 0.01-0.9 mg/ml, 0.01-0.8 mg/ml, 0.01-0.7mg/ml, 0.01-0.6 mg/ml, 0.01-0.5 mg/ml, 0.01-0.4 mg/ml, 0.01-0.3 mg/ml,0.01-0.2 mg/ml, 0.01-0.1 mg/ml, 0.05-1.0 mg/ml, 0.05-0.9 mg/ml, 0.05-0.8mg/ml, 0.05-0.7 mg/ml, 0.05-0.6 mg/ml, 0.05-0.5 mg/ml, 0.05-0.4 mg/ml,0.05-0.3 mg/ml, 0.05-0.2 mg/ml, 0.05-0.1 mg/ml, 0.1-1.0 mg/ml, 0.2-0.9mg/ml, 0.3-0.8 mg/ml, 0.4-0.7 mg/ml, or 0.5-0.6 mg/ml.

Typically, a suitable RNA solution may also contain a buffering agentand/or salt. Generally, buffering agents can include HEPES, Tris,ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate,potassium phosphate or sodium phosphate. In some embodiments, suitableconcentration of the buffering agent may be in a range from about 0.1 mMto 100 mM, 0.5 mM to 90 mM, 1.0 mM to 80 mM, 2 mM to 70 mM, 3 mM to 60mM, 4 mM to 50 mM, 5 mM to 40 mM, 6 mM to 30 mM, 7 mM to 20 mM, 8 mM to15 mM, or 9 to 12 mM.

Exemplary salts can include sodium chloride, magnesium chloride, andpotassium chloride. In some embodiments, suitable concentration of saltsin an RNA solution may be in a range from about 1 mM to 500 mM, 5 mM to400 mM, 10 mM to 350 mM, 15 mM to 300 mM, 20 mM to 250 mM, 30 mM to 200mM, 40 mM to 190 mM, 50 mM to 180 mM, 50 mM to 170 mM, 50 mM to 160 mM,50 mM to 150 mM, or 50 mM to 100 mM.

In some embodiments, a suitable RNA solution may have a pH in a rangefrom about 3.5-6.5, 3.5-6.0, 3.5-5.5, 3.5-5.0, 3.5-4.5, 4.0-5.5,4.0-5.0, 4.0-4.9, 4.0-4.8, 4.0-4.7, 4.0-4.6, or 4.0-4.5.

Various methods may be used to prepare an RNA solution suitable for thepresent invention. In some embodiments, RNA may be directly dissolved ina buffer solution described herein. In some embodiments, an RNA solutionmay be generated by mixing an RNA stock solution with a buffer solutionprior to mixing with a lipid solution for encapsulation. In someembodiments, an RNA solution may be generated by mixing an RNA stocksolution with a buffer solution immediately before mixing with a lipidsolution for encapsulation.

According to the present invention, a lipid solution contains a mixtureof lipids suitable to form transfer vehicles for encapsulation of RNA.In some embodiments, a suitable lipid solution is ethanol based. Forexample, a suitable lipid solution may contain a mixture of desiredlipids dissolved in pure ethanol (i.e. 100% ethanol). In anotherembodiment, a suitable lipid solution is isopropyl alcohol based. Inanother embodiment, a suitable lipid solution isdimethylsulfoxide-based. In another embodiment, a suitable lipidsolution is a mixture of suitable solvents including, but not limitedto, ethanol, isopropyl alcohol and dimethylsulfoxide.

A suitable lipid solution may contain a mixture of desired lipids atvarious concentrations. In some embodiments, a suitable lipid solutionmay contain a mixture of desired lipids at a total concentration in arange from about 0.1-100 mg/ml, 0.5-90 mg/ml, 1.0-80 mg/ml, 1.0-70mg/ml, 1.0-60 mg/ml, 1.0-50 mg/ml, 1.0-40 mg/ml, 1.0-30 mg/ml, 1.0-20mg/ml, 1.0-15 mg/ml, 1.0-10 mg/ml, 1.0-9 mg/ml, 1.0-8 mg/ml, 1.0-7mg/ml, 1.0-6 mg/ml, or 1.0-5 mg/ml.

11. Targeting

The present invention also contemplates the discriminatory targeting oftarget cells and tissues by both passive and active targeting means. Thephenomenon of passive targeting exploits the natural distributionspatterns of a transfer vehicle in vivo without relying upon the use ofadditional excipients or means to enhance recognition of the transfervehicle by target cells. For example, transfer vehicles which aresubject to phagocytosis by the cells of the reticulo-endothelial systemare likely to accumulate in the liver or spleen, and accordingly mayprovide a means to passively direct the delivery of the compositions tosuch target cells.

Alternatively, the present invention contemplates active targeting,which involves the use of targeting moieties that may be bound (eithercovalently or non-covalently) to the transfer vehicle to encouragelocalization of such transfer vehicle at certain target cells or targettissues. For example, targeting may be mediated by the inclusion of oneor more endogenous targeting moieties in or on the transfer vehicle toencourage distribution to the target cells or tissues. Recognition ofthe targeting moiety by the target tissues actively facilitates tissuedistribution and cellular uptake of the transfer vehicle and/or itscontents in the target cells and tissues (e.g., the inclusion of anapolipoprotein-E targeting ligand in or on the transfer vehicleencourages recognition and binding of the transfer vehicle to endogenouslow density lipoprotein receptors expressed by hepatocytes). As providedherein, the composition can comprise a moiety capable of enhancingaffinity of the composition to the target cell. Targeting moieties maybe linked to the outer bilayer of the lipid particle during formulationor post-formulation. These methods are well known in the art. Inaddition, some lipid particle formulations may employ fusogenic polymerssuch as PEAA, hemagluttinin, other lipopeptides (see U.S. patentapplication Ser. No. 08/835,281, and 60/083,294, which are incorporatedherein by reference) and other features useful for in vivo and/orintracellular delivery. In other some embodiments, the compositions ofthe present invention demonstrate improved transfection efficacies,and/or demonstrate enhanced selectivity towards target cells or tissuesof interest. Contemplated therefore are compositions which comprise oneor more moieties (e.g., peptides, aptamers, oligonucleotides, a vitaminor other molecules) that are capable of enhancing the affinity of thecompositions and their nucleic acid contents for the target cells ortissues. Suitable moieties may optionally be bound or linked to thesurface of the transfer vehicle. In some embodiments, the targetingmoiety may span the surface of a transfer vehicle or be encapsulatedwithin the transfer vehicle. Suitable moieties and are selected basedupon their physical, chemical or biological properties (e.g., selectiveaffinity and/or recognition of target cell surface markers or features).Cell-specific target sites and their corresponding targeting ligand canvary widely. Suitable targeting moieties are selected such that theunique characteristics of a target cell are exploited, thus allowing thecomposition to discriminate between target and non-target cells. Forexample, compositions of the invention may include surface markers(e.g., apolipoprotein-B or apolipoprotein-E) that selectively enhancerecognition of, or affinity to hepatocytes (e.g., by receptor-mediatedrecognition of and binding to such surface markers). As an example, theuse of galactose as a targeting moiety would be expected to direct thecompositions of the present invention to parenchymal hepatocytes, oralternatively the use of mannose containing sugar residues as atargeting ligand would be expected to direct the compositions of thepresent invention to liver endothelial cells (e.g., mannose containingsugar residues that may bind preferentially to the asialoglycoproteinreceptor present in hepatocytes). (See Hillery A M, et al. “DrugDelivery and Targeting: For Pharmacists and Pharmaceutical Scientists”(2002) Taylor & Francis, Inc.) The presentation of such targetingmoieties that have been conjugated to moieties present in the transfervehicle (e.g., a lipid nanoparticle) therefore facilitate recognitionand uptake of the compositions of the present invention in target cellsand tissues. Examples of suitable targeting moieties include one or morepeptides, proteins, aptamers, vitamins and oligonucleotides.

In particular embodiments, a transfer vehicle comprises a targetingmoiety. In some embodiments, the targeting moiety mediatesreceptor-mediated endocytosis selectively into a specific population ofcells. In some embodiments, the targeting moiety is capable of bindingto a T cell antigen. In some embodiments, the targeting moiety iscapable of binding to a NK, NKT, or macrophage antigen. In someembodiments, the targeting moiety is capable of binding to a proteinselected from the group CD3, CD4, CD8, PD-1, 4-1BB, and CD2. In someembodiments, the targeting moiety is an single chain Fv (scFv) fragment,nanobody, peptide, peptide-based macrocycle, minibody, heavy chainvariable region, light chain variable region or fragment thereof. Insome embodiments, the targeting moiety is selected from T-cell receptormotif antibodies, T-cell α chain antibodies, T-cell β chain antibodies,T-cell γ chain antibodies, T-cell δ chain antibodies, CCR7 antibodies,CD3 antibodies, CD4 antibodies, CD5 antibodies, CD7 antibodies, CD8antibodies, CD11b antibodies, CD11c antibodies, CD16 antibodies, CD19antibodies, CD20 antibodies, CD21 antibodies, CD22 antibodies, CD25antibodies, CD28 antibodies, CD34 antibodies, CD35 antibodies, CD40antibodies, CD45RA antibodies, CD45RO antibodies, CD52 antibodies, CD56antibodies, CD62L antibodies, CD68 antibodies, CD80 antibodies, CD95antibodies, CD117 antibodies, CD127 antibodies, CD133 antibodies, CD137(4-1BB) antibodies, CD163 antibodies, F4/80 antibodies, IL-4Rαantibodies, Sca-1 antibodies, CTLA-4 antibodies, GITR antibodies GARPantibodies, LAP antibodies, granzyme B antibodies, LFA-1 antibodies,transferrin receptor antibodies, and fragments thereof. In someembodiments, the targeting moiety is a small molecule binder of anectoenzyme on lymphocytes. Small molecule binders of ectoenzymes includeA2A inhibitors CD73 inhibitors, CD39 or adesines receptors A2aR andA2bR. Potential small molecules include AB928.

In some embodiments, transfer vehicles are formulated and/or targeted asdescribed in Shobaki N, Sato Y, Harashima H. Mixing lipids to manipulatethe ionization status of lipid nanoparticles for specific tissuetargeting. Int J Nanomedicine. 2018; 13:8395-8410. Published 2018 Dec.10. In some embodiments, a transfer vehicle is made up of 3 lipid types.In some embodiments, a transfer vehicle is made up of 4 lipid types. Insome embodiments, a transfer vehicle is made up of 5 lipid types. Insome embodiments, a transfer vehicle is made up of 6 lipid types.

12. Target Cells

Where it is desired to deliver a nucleic acid to an immune cell, theimmune cell represents the target cell. In some embodiments, thecompositions of the invention transfect the target cells on adiscriminatory basis (i.e., do not transfect non-target cells). Thecompositions of the invention may also be prepared to preferentiallytarget a variety of target cells, which include, but are not limited to,T cells, B cells, macrophages, and dentritic cells.

In some embodiments, the target cells are deficient in a protein orenzyme of interest. For example, where it is desired to deliver anucleic acid to a hepatocyte, the hepatocyte represents the target cell.In some embodiments, the compositions of the invention transfect thetarget cells on a discriminatory basis (i.e., do not transfectnon-target cells). The compositions of the invention may also beprepared to preferentially target a variety of target cells, whichinclude, but are not limited to, hepatocytes, epithelial cells,hematopoietic cells, epithelial cells, endothelial cells, lung cells,bone cells, stem cells, mesenchymal cells, neural cells (e.g., meninges,astrocytes, motor neurons, cells of the dorsal root ganglia and anteriorhorn motor neurons), photoreceptor cells (e.g., rods and cones), retinalpigmented epithelial cells, secretory cells, cardiac cells, adipocytes,vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells,beta cells, pituitary cells, synovial lining cells, ovarian cells,testicular cells, fibroblasts, B cells, T cells, reticulocytes,leukocytes, granulocytes and tumor cells.

The compositions of the invention may be prepared to preferentiallydistribute to target cells such as in the heart, lungs, kidneys, liver,and spleen. In some embodiments, the compositions of the inventiondistribute into the cells of the liver or spleen to facilitate thedelivery and the subsequent expression of the circRNA comprised thereinby the cells of the liver (e.g., hepatocytes) or the cells of spleen(e.g., immune cells). The targeted cells may function as a biological“reservoir” or “depot” capable of producing, and systemically excretinga functional protein or enzyme. Accordingly, in one embodiment of theinvention the transfer vehicle may target hepatocytes or immune cellsand/or preferentially distribute to the cells of the liver or spleenupon delivery. In an embodiment, following transfection of the targethepatocytes or immune cells, the circRNA loaded in the vehicle aretranslated and a functional protein product is produced, excreted andsystemically distributed. In other embodiments, cells other thanhepatocytes (e.g., lung, spleen, heart, ocular, or cells of the centralnervous system) can serve as a depot location for protein production.

In one embodiment, the compositions of the invention facilitate asubject's endogenous production of one or more functional proteinsand/or enzymes. In an embodiment of the present invention, the transfervehicles comprise circRNA which encode a deficient protein or enzyme.Upon distribution of such compositions to the target tissues and thesubsequent transfection of such target cells, the exogenous circRNAloaded into the transfer vehicle (e.g., a lipid nanoparticle) may betranslated in vivo to produce a functional protein or enzyme encoded bythe exogenously administered circRNA (e.g., a protein or enzyme in whichthe subject is deficient). Accordingly, the compositions of the presentinvention exploit a subject's ability to translate exogenously- orrecombinantly-prepared circRNA to produce an endogenously-translatedprotein or enzyme, and thereby produce (and where applicable excrete) afunctional protein or enzyme. The expressed or translated proteins orenzymes may also be characterized by the in vivo inclusion of nativepost-translational modifications which may often be absent inrecombinantly-prepared proteins or enzymes, thereby further reducing theimmunogenicity of the translated protein or enzyme.

The administration of circRNA encoding a deficient protein or enzymeavoids the need to deliver the nucleic acids to specific organelleswithin a target cell. Rather, upon transfection of a target cell anddelivery of the nucleic acids to the cytoplasm of the target cell, thecircRNA contents of a transfer vehicle may be translated and afunctional protein or enzyme expressed.

In some embodiments, a circular RNA comprises one or more miRNA bindingsites. In some embodiments, a circular RNA comprises one or more miRNAbinding sites recognized by miRNA present in one or more non-targetcells or non-target cell types (e.g., Kupffer cells or hepatic cells)and not present in one or more target cells or target cell types (e.g.,hepatocytes or T cells). In some embodiments, a circular RNA comprisesone or more miRNA binding sites recognized by miRNA present in anincreased concentration in one or more non-target cells or non-targetcell types (e.g., Kupffer cells or hepatic cells) compared to one ormore target cells or target cell types (e.g., hepatocytes or T cells).miRNAs are thought to function by pairing with complementary sequenceswithin RNA molecules, resulting in gene silencing.

13. Pharmaceutical Compositions

In certain embodiments, provided herein are compositions (e.g.,pharmaceutical compositions) comprising a therapeutic agent providedherein. In some embodiments, the therapeutic agent is a circular RNApolynucleotide provided herein. In some embodiments the therapeuticagent is a vector provided herein. In some embodiments, the therapeuticagent is a cell comprising a circular RNA or vector provided herein(e.g., a human cell, such as a human T cell). In certain embodiments,the composition further comprises a pharmaceutically acceptable carrier.In some embodiments, the compositions provided herein comprise atherapeutic agent provided herein in combination with otherpharmaceutically active agents or drugs, such as anti-inflammatory drugsor antibodies capable of targeting B cell antigens, e.g., anti-CD20antibodies, e.g., rituximab.

With respect to pharmaceutical compositions, the pharmaceuticallyacceptable carrier can be any of those conventionally used and islimited only by chemico-physical considerations, such as solubility andlack of reactivity with the active agent(s), and by the route ofadministration. The pharmaceutically acceptable carriers describedherein, for example, vehicles, adjuvants, excipients, and diluents, arewell-known to those skilled in the art and are readily available to thepublic. It is preferred that the pharmaceutically acceptable carrier beone which is chemically inert to the therapeutic agent(s) and one whichhas no detrimental side effects or toxicity under the conditions of use.

The choice of carrier will be determined in part by the particulartherapeutic agent, as well as by the particular method used toadminister the therapeutic agent. Accordingly, there are a variety ofsuitable formulations of the pharmaceutical compositions providedherein.

In certain embodiments, the pharmaceutical composition comprises apreservative. In certain embodiments, suitable preservatives mayinclude, for example, methylparaben, propylparaben, sodium benzoate, andbenzalkonium chloride. Optionally, a mixture of two or morepreservatives may be used. The preservative or mixtures thereof aretypically present in an amount of about 0.0001% to about 2% by weight ofthe total composition.

In some embodiments, the pharmaceutical composition comprises abuffering agent. In some embodiments, suitable buffering agents mayinclude, for example, citric acid, sodium citrate, phosphoric acid,potassium phosphate, and various other acids and salts. A mixture of twoor more buffering agents optionally may be used. The buffering agent ormixtures thereof are typically present in an amount of about 0.001% toabout 4% by weight of the total composition.

In some embodiments, the concentration of therapeutic agent in thepharmaceutical composition can vary, e.g., less than about 1%, or atleast about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, or about 50% or more by weight, and can be selectedprimarily by fluid volumes, and viscosities, in accordance with theparticular mode of administration selected.

The following formulations for oral, aerosol, parenteral (e.g.,subcutaneous, intravenous, intraarterial, intramuscular, intradermal,intraperitoneal, and intrathecal), and topical administration are merelyexemplary and are in no way limiting. More than one route can be used toadminister the therapeutic agents provided herein, and in certaininstances, a particular route can provide a more immediate and moreeffective response than another route.

Formulations suitable for oral administration can comprise or consist of(a) liquid solutions, such as an effective amount of the therapeuticagent dissolved in diluents, such as water, saline, or orange juice; (b)capsules, sachets, tablets, lozenges, and troches, each containing apredetermined amount of the active ingredient, as solids or granules;(c) powders; (d) suspensions in an appropriate liquid; and (e) suitableemulsions. Liquid formulations may include diluents, such as water andalcohols, for example, ethanol, benzyl alcohol and the polyethylenealcohols, either with or without the addition of a pharmaceuticallyacceptable surfactant. Capsule forms can be of the ordinary hard or softshelled gelatin type containing, for example, surfactants, lubricants,and inert fillers, such as lactose, sucrose, calcium phosphate, and cornstarch. Tablet forms can include one or more of lactose, sucrose,mannitol, corn starch, potato starch, alginic acid, microcrystallinecellulose, acacia, gelatin, guar gum, colloidal silicon dioxide,croscarmellose sodium, talc, magnesium stearate, calcium stearate, zincstearate, stearic acid, and other excipients, colorants, diluents,buffering agents, disintegrating agents, moistening agents,preservatives, flavoring agents, and other pharmacologically compatibleexcipients. Lozenge forms can comprise the therapeutic agent with aflavorant, usually sucrose, acacia or tragacanth. Pastilles can comprisethe therapeutic agent with an inert base, such as gelatin and glycerin,or sucrose and acacia, emulsions, gels, and the like containing, inaddition to, such excipients as are known in the art.

Formulations suitable for parenteral administration include aqueous andnonaqueous isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and nonaqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.In some embodiments, the therapeutic agents provided herein can beadministered in a physiologically acceptable diluent in a pharmaceuticalcarrier, such as a sterile liquid or mixture of liquids, includingwater, saline, aqueous dextrose and related sugar solutions, an alcoholsuch as ethanol or hexadecyl alcohol, a glycol such as propylene glycolor polyethylene glycol, dimethylsulfoxide, glycerol, ketals such as2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, poly(ethyleneglycol) 400,oils, fatty acids, fatty acid esters or glycerides, or acetylated fattyacid glycerides with or without the addition of a pharmaceuticallyacceptable surfactant such as a soap or a detergent, suspending agentsuch as pectin, carbomers, methylcellulose,hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifyingagents and other pharmaceutical adjuvants.

Oils, which can be used in parenteral formulations in some embodiments,include petroleum, animal oils, vegetable oils, or synthetic oils.Specific examples of oils include peanut, soybean, sesame, cottonseed,corn, olive, petrolatum, and mineral oil. Suitable fatty acids for usein parenteral formulations include oleic acid, stearic acid, andisostearic acid. Ethyl oleate and isopropyl myristate are examples ofsuitable fatty acid esters.

Suitable soaps for use in certain embodiments of parenteral formulationsinclude fatty alkali metal, ammonium, and triethanolamme salts, andsuitable detergents include (a) cationic detergents such as, forexample, dimethyl dialkyl ammonium halides. and alkyl pyridiniumhalides, (b) anionic detergents such as, for example, alkyl, aryl, andolefin sulfonates, alky, olefin, ether, and monoglyceride sulfates, andsulfosuccinates, (c) nonionic detergents such as, for example, fattyamine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylenecopolymers, (d) amphoteric detergents such as, for example,alkyl-β-aminopropionates, and 2-alkyl-imidazoline quaternary ammoniumsalts, and (e) mixtures thereof.

In some embodiments, the parenteral formulations will contain, forexample, from about 0.5% to about 25% by weight of the therapeutic agentin solution. Preservatives and buffers may be used. In order to minimizeor eliminate irritation at the site of injection, such compositions maycontain one or more nonionic surfactants having, for example, ahydrophile-lipophile balance (HLB) of from about 12 to about 17. Thequantity of surfactant in such formulations will typically range, forexample, from about 5% to about 15% by weight. Suitable surfactantsinclude polyethylene glycol sorbitan fatty acid esters, such as sorbitanmonooleate and high molecular weight adducts of ethylene oxide with ahydrophobic base, formed by the condensation of propylene oxide withpropylene glycol. The parenteral formulations can be presented inunit-dose or multi-dose sealed containers, such as ampoules or vials,and can be stored in a freeze-dried (lyophilized) condition requiringonly the addition of a sterile liquid excipient, for example, water, forinjections, immediately prior to use. Extemporaneous injection solutionsand suspensions can be prepared from sterile powders, granules, andtablets of the kind previously described.

In certain embodiments, injectable formulations are provided herein. Therequirements for effective pharmaceutical carriers for injectablecompositions are well-known to those of ordinary skill in the art (see,e.g., Pharmaceutics and Pharmacy Practice, J. B. Lippincott Company,Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), andASHP Handbook on Injectable Drugs, Toissel, 4th ed, pages 622-630(1986)).

In some embodiments, topical formulations are provided herein. Topicalformulations, including those that are useful for transdermal drugrelease, are suitable in the context of certain embodiments providedherein for application to skin. In some embodiments, the therapeuticagent alone or in combination with other suitable components, can bemade into aerosol formulations to be administered via inhalation. Theseaerosol formulations can be placed into pressurized acceptablepropellants, such as dichlorodifluoromethane, propane, nitrogen, and thelike. They also may be formulated as pharmaceuticals for non-pressuredpreparations, such as in a nebulizer or an atomizer. Such sprayformulations also may be used to spray mucosa.

In certain embodiments, the therapeutic agents provided herein can beformulated as inclusion complexes, such as cyclodextrin inclusioncomplexes, or liposomes. Liposomes can serve to target the therapeuticagents to a particular tissue. Liposomes also can be used to increasethe half-life of the therapeutic agents. Many methods are available forpreparing liposomes, as described in, for example, Szoka et al., Ann.Rev. Biophys. Bioeng., 9, 467 (1980) and U.S. Pat. Nos. 4,235,871,4,501,728, 4,837,028, and 5,019,369.

In some embodiments, the therapeutic agents provided herein areformulated in time-released, delayed release, or sustained releasedelivery systems such that the delivery of the composition occurs priorto, and with sufficient time to cause, sensitization of the site to betreated. Such systems can avoid repeated administrations of thetherapeutic agent, thereby increasing convenience to the subject and thephysician, and may be particularly suitable for certain compositionembodiments provided herein. In one embodiment, the compositions of theinvention are formulated such that they are suitable forextended-release of the circRNA contained therein. Such extended-releasecompositions may be conveniently administered to a subject at extendeddosing intervals. For example, in one embodiment, the compositions ofthe present invention are administered to a subject twice day, daily orevery other day. In an embodiment, the compositions of the presentinvention are administered to a subject twice a week, once a week, everyten days, every two weeks, every three weeks, every four weeks, once amonth, every six weeks, every eight weeks, every three months, everyfour months, every six months, every eight months, every nine months orannually.

In some embodiments, a protein encoded by an inventive polynucleotide isproduced by a target cell for sustained amounts of time. For example,the protein may be produced for more than one hour, more than four, morethan six, more than 12, more than 24, more than 48 hours, or more than72 hours after administration. In some embodiments the polypeptide isexpressed at a peak level about six hours after administration. In someembodiments the expression of the polypeptide is sustained at least at atherapeutic level. In some embodiments the polypeptide is expressed atleast at a therapeutic level for more than one, more than four, morethan six, more than 12, more than 24, more than 48, or more than 72hours after administration. In some embodiments, the polypeptide isdetectable at a therapeutic level in patient serum or tissue (e.g.,liver or lung). In some embodiments, the level of detectable polypeptideis from continuous expression from the circRNA composition over periodsof time of more than one, more than four, more than six, more than 12,more than 24, more than 48, or more than 72 hours after administration.

In certain embodiments, a protein encoded by an inventive polynucleotideis produced at levels above normal physiological levels. The level ofprotein may be increased as compared to a control. In some embodiments,the control is the baseline physiological level of the polypeptide in anormal individual or in a population of normal individuals. In otherembodiments, the control is the baseline physiological level of thepolypeptide in an individual having a deficiency in the relevant proteinor polypeptide or in a population of individuals having a deficiency inthe relevant protein or polypeptide. In some embodiments the control canbe the normal level of the relevant protein or polypeptide in theindividual to whom the composition is administered. In other embodimentsthe control is the expression level of the polypeptide upon othertherapeutic intervention, e.g., upon direct injection of thecorresponding polypeptide, at one or more comparable time points.

In certain embodiments, the levels of a protein encoded by an inventivepolynucleotide are detectable at 3 days, 4 days, 5 days, or 1 week ormore after administration. Increased levels of secreted protein may beobserved in the serum and/or in a tissue (e.g., liver or lung).

In some embodiments, the method yields a sustained circulation half-lifeof a protein encoded by an inventive polynucleotide. For example, theprotein may be detected for hours or days longer than the half-lifeobserved via subcutaneous injection of the protein or mRNA encoding theprotein. In some embodiments, the half-life of the protein is 1 day, 2days, 3 days, 4 days, 5 days, or 1 week or more.

Many types of release delivery systems are available and known to thoseof ordinary skill in the art. They include polymer based systems such aspoly(lactide-glycolide), copolyoxalates, polycaprolactones,polyesteramides, polyorthoesters, polyhydroxybutyric acid, andpolyanhydrides. Microcapsules of the foregoing polymers containing drugsare described in, for example, U.S. Pat. No. 5,075,109. Delivery systemsalso include non-polymer systems that are lipids including sterols suchas cholesterol, cholesterol esters, and fatty acids or neutral fats suchas mono-di- and tri-glycerides; hydrogel release systems; sylasticsystems; peptide based systems: wax coatings; compressed tablets usingconventional binders and excipients; partially fused implants; and thelike. Specific examples include, but are not limited to: (a) erosionalsystems in which the active composition is contained in a form within amatrix such as those described in U.S. Pat. Nos. 4,452,775, 4,667,014,4,748,034, and 5,239,660 and (b) diffusional systems in which an activecomponent permeates at a controlled rate from a polymer such asdescribed in U.S. Pat. Nos. 3,832,253 and 3,854,480. In addition,pump-based hardware delivery systems can be used, some of which areadapted for implantation.

In some embodiments, the therapeutic agent can be conjugated eitherdirectly or indirectly through a linking moiety to a targeting moiety.Methods for conjugating therapeutic agents to targeting moieties isknown in the art. See, for instance, Wadwa et al., J, Drug Targeting3:111 (1995) and U.S. Pat. No. 5,087,616.

In some embodiments, the therapeutic agents provided herein areformulated into a depot form, such that the manner in which thetherapeutic agent is released into the body to which it is administeredis controlled with respect to time and location within the body (see,for example, U.S. Pat. No. 4,450,150). Depot forms of therapeutic agentscan be, for example, an implantable composition comprising thetherapeutic agents and a porous or non-porous material, such as apolymer, wherein the therapeutic agents are encapsulated by or diffusedthroughout the material and/or degradation of the non-porous material.The depot is then implanted into the desired location within the bodyand the therapeutic agents are released from the implant at apredetermined rate.

14. Therapeutic Methods

In certain aspects, provided herein is a method of treating and/orpreventing a condition, e.g., cancer.

In certain embodiments, the therapeutic agents provided herein arecoadministered with one or more additional therapeutic agents (e.g., inthe same pharmaceutical composition or in separate pharmaceuticalcompositions). In some embodiments, the therapeutic agent providedherein can be administered first and the one or more additionaltherapeutic agents can be administered second, or vice versa.Alternatively, the therapeutic agent provided herein and the one or moreadditional therapeutic agents can be administered simultaneously.

In some embodiments, the subject is a mammal. In some embodiments, themammal referred to herein can be any mammal, including, but not limitedto, mammals of the order Rodentia, such as mice and hamsters, or mammalsof the order Logomorpha, such as rabbits. The mammals may be from theorder Carnivora, including Felines (cats) and Canines (dogs). Themammals may be from the order Artiodactyla, including Bovines (cows) andSwines (pigs), or of the order Perssodactyla, including Equines(horses). The mammals may be of the order Primates, Ceboids, or Simoids(monkeys) or of the order Anthropoids (humans and apes). Preferably, themammal is a human.

15. Sequences

TABLE 17 IRES sequences. SEQ ID NO IRES Sequence   1 EMCV-Acccccctctccctccccccctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccatattgccgtcttttggcaatgtgagggcccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaaggaagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgacaggtgcctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctctcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtgcacatgctttacatgtgtttagtcgaggttaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaacacgatgataatatggccacaacc   2 EMCV-Bctccccctccccccccttactatactggccgaagccacttggaataaggccggtgtgcgtttgtctacatgctattttctaccgcattaccgtcttatggtaatgtgagggtccagaacctgaccctgtcttcttgacgaacactcctaggggtctttcccctctcgacaaaggagtgtaaggtctgttgaatgtcgtgaaggaagcagttcctctggaagcttcttaaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggtgacaggtgcctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctctcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtgcacgtgctttacacgtgttgagtcgaggtgaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaccacgattacaat   3 EMCV-Bfttgccagtctgctcgatatcgcaggctgggtccgtgactacccactccccctttcaacgtgaaggctacgatagtgccagggcgggtactgccgtaagtgccaccccaaacaacaacaacaaaacaaactccccctccccccccttactatactggccgaagccacttggaataaggccggtgtgcgtttgtctacatgctattttctaccgcattaccgtcttatggtaatgtgagggtccagaacctgaccctgtcttcttgacgaacactcctaggggtctttcccctctcgacaaaggagtgtaaggtctgttgaatgtcgtgaaggaagcagttcctctggaagcttcttaaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggtgacaggtgcctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctctcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtgcacgtgctttacacgtgttgagtcgaggtgaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaccacgattacaat   4 EMCV-CfttgccagtctgctcgatatcgcaggctgggtccgtgactacccactccccctttcaacgtgaaggctacgatagtgccagggcgggtactgccgtaagtgccaccccaaaacaacaacaaccccccctctccctccTccccccctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccatattgccgtcttttggcaatgtgagggcccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtctgagaatgtcgtgaaggaagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgacaggtgcctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctctcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtgcacatgctttacatgtgtttagtcgaggttaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaacacgatgataat   5 EMCV pEC9ccccccccctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccatattgccgtcttttggcaatgtgagggcccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaaggaagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgacaggtgcctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctctcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtgcacatgctttacatgtgtttagtcgaggttaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaacacgatgataat   6 Picobir-gtaaattaaatgctatttacaaaatttaaacagaaaggagagatgttatgaaccggttttacaaggtttnaviruscatacatcgaaaatagcactacctggggcagccgacacactaacatcgtctgtttaaccagaagtgttactgaaaggaggttattta   7 HCV QC64acctgcccctaataggggcgacactccgccatgaatcactcccctgtgaggaactactgtcttcacgcagaaagcgtctagccatggcgttagtatgagtgtcgtacagcctccaggcccccccctcccgggagagccatagtggtctgcggaaccggtgagtacaccggaattgccgggaagactgggtcctttcttggataaacccactctatgcccggacatttgggcgtgcccccgcaagactgctagccgagtagcgttgggttgcgaaaggccttgtggtactgcctgatagggtgcttgcgagtgccccgggaggtctcgtagaccgtgcatc   8 HumanctacaagctttgtgtaaacaaacttttgtttggcttttctcaagcttctctcacatcaggccccaaagatCosavirusgtcctgaaggtaccccgtgtatctgaggatgagcaccatcgactacccggacctgcaaaattttgc E/Daaacgcatgtggtatcccagccccctcctctcggggagggggctttgctcactcagcacaggatctgatcaggagatccacctccggtgctttacaccggggcgtggatttaaaaattgcccaaggcctggcgcacaacctaggggactaggttttccttatattttaaagctgtcaat   9 HumangtcttaggacgacgcatgtggtatcccagcccccgcctacattggcgggggcttttgaagcaccaCosavirusgacactggatctgatcaggaggagggtagctgctttacagcccctcttaaaaattgcccaaggtcc Fggccacccaacctaggggactaggttttccttttatttttaaattgtcatt  10 HumanacatgggggagactgcatgtggcagtcttgaaacgtgtggtttgacgtctaccttatatggcagtggCosavirusgtggagtactgcaaagatgtcaccgtgattacacggtttttgaaccccacaccggctgtttgacgct JMYcgtagggcagcaggtttattttcattaaaattcttactttctagctgcatgagttctattcatgcagacggagtgatactcccgttccttcttggacaggttgcctccacgccctttgtggatcttaaggtgaccaagtcactggtgttggaggtgaagatagagagtcctcttgggaatgtcatgtggctgtgccaggggttgtagcgatgccattcgtgtgtgcggatttcctctcgtggtgacacgagcctcacaggccaaaagccccgtccgaaaggacccgaatggtggagtgaccctgactcccccctgcatagttttgtgattaggaacttgaggaatttctgtcataaatctctatcacatcaggccccaaagatgtcctgaaggtaccctgtgtatctgaggatgagcaccaccgactacccggacttgcattagcagacacatgtggttgcccagccccacctcttcagaggtggggctttgctcactcagcacaggatctgatcaggagccccgctcgtgtgctttacactcgacgcggggttaaaaattgcccaaggcctggcacaacaacctaggggactaggttttcctatttttgtaaattatgtcaat  11 Rhino-gtgacaatcagccagattgttaacggtcaagcacttctgtttccccggtacccttgtatacgcttcaccvirus cgaggcgaaaagtgaggttatcgttatccgcaaagtgcctacgagaagcctagtagcacttttgaaNAT001gcctatggctggtcgctcaactgtttacccagcagtagacctggcagatgaggctagatgttccccaccagcgatggtgatctagcctgcgtggctgcctgcacactctattgagtgtgaagccagaaagtggacaaggtgtgaagagcctattgtgctcactttgagtcctccggcccctgaatgtggctaatcctaaccccgtagctgttgcatgtaatccaacatgtctgcagtcgtaatgggcaactatgggatggaaccaactactttgggtgtccgtgtttcttgtttttctttatgcttgcttatggtgacaactgtagttattacatttgttacc  12 HRV14ttaaaacagcggatgggtatcccaccattcgacccattgggtgtagtactctggtactatgtacctttgtacgcctgtttctccccaaccacccttccttaaaattcccacccatgaaacgttagaagcttgacattaaagtacaataggtggcgccatatccaatggtgtctatgtacaagcacttctgtttcccaggagcgaggtataggctgtacccactgccaaaagcctttaaccgttatccgccaaccaactacgtaacagttagtaccatcttgttcttgactggacgttcgatcaggtggattttccctccactagtttggtcgatgaggctaggaattccccacgggtgaccgtgtcctagcctgcgtggcggccaacccagcttatgctgggacgcccttttaaggacatggtgtgaagactcgcatgtgcttggttgtgagtcctccggcccctgaatgcggctaaccttaaccctagagccttatgccacgatccagtggagtaaggtcgtaatgagcaattccgggacgggaccgactactttgggtgtccgtgtttctcatttttcttcatattgtcttatggtcacagcatatatatacatatactgtgatc  13 HRV89ttaaaactgggagtgggttgttcccactcactccacccatgcggtgttgtactctgttattacggtaactttgtacgccagtttttcccacccttccccataatgtaacttagaagtttgtacaatatgaccaataggtgacaatcatccagactgtcaaaggtcaagcacttctgtttccccggtcaatgaggatatgctttacccaaggcaaaaaccttagagatcgttatccccacactgcctacacagagcccagtaccatttttgatataattgggttggtcgctccctgcaaacccagcagtagacctggcagatgaggctggacattccccactggcgacagtggtccagcctgcgtggctgcctgctcacccttcttgggtgagaagcctaattattgacaaggtgtgaagagccgcgtgtgctcagtgtgcttcctccggcccctgaatgtggctaaccttaaccctgcagccgttgcccataatccaatgggtttgcggtcgtaatgcgtaagtgcgggatgggaccaactactttgggtgtccgtgtttcctgtttttcttttgattgcattttatggtgacaatttatagtgtatagattgtcatc  14 HRVC-02ttaaaactgggtacaggttgttcccacctgtatcacccacgtggtgtggtgctcttgtattccggtacacttgcacgccagtttgccacccctcacccgtcgtaacttagaagctaacaactcgaccaacaggcggtggtaaaccataccacttacggtcaagcactcctgtttccccggtatgcgaggaatagactcctacagggttgaagcctcaagtatcgttatccgcattggtactacgcaaagcttagtagtgccttgaaagtcccttggttggtcgctccgctagtttcccctagtagacctggcagatgaggcaggacactccccactggcgacagtggtcctgcctgcgtggctgcctgcgcacccttaggggtgcgaagccaagtgacagacaaggtgtgaagagccccgtgtgctaccaatgagtcctccggcccctgaatgcggctaatccaaccccacagctattgcacacaagccagtgtgtatgtagtcgtaatgagcaattgtgggacggaaccgactactttgggtgtccgtgtttccttttattcttatcattctgcttatggtgacaatactgtgaaatagtgttgttacc  15 HRV-A21taaaactggatccaggttgttcccacctggatctcctattgggagttgtactctattattccggtaattttgtacgccagttttatcttccccctccccaattgtaacttagaaggttatcaatacgaccaataggtggtagttagccaaactaccaaaggtcaagcacttctgtttccccggtcaaagttgatatgctccaacagggcaaaaacaactgagatcgttatccgcaaagtgcctacgcaaagcctagtaacacctttgaagatttatggttggtcgttccgctatttcccatagtagacctggcagatgaggctagaaatcccccactggcgacagtgctctagcctgcgtggctgcctgcgcaccccttgggtgcgaagccatacattggacaaggtgtgaagagccccgtgtgctcactttgagtcctccggcccctgaatgtggctaaccttaaccctgcagctagtgcatgtaatccaacatgttgctagtcgtaatgagtaattgcgggacgggaccaactactttgggtgtccgtgtttcactttttccttttaatattgcttatggtgacaatatatatagctatatatattgacacc  16 SalivirusttcccctgcaaccattacgcttactcgcatgtgcattgagtggtgcatgtgttgaacaaacagctacaA SH1ctcacatgggggcgggttttcccgccctacggcttctcgcgaggcccacccctcccctttctcccataactacagtgctttggtaggtaagcatcctgatcccccgcggaagctgctcacgtggcaactgtggggacccagacaggttatcaaaggcacccggtctttccgccttcaggagtatccctgctagcgaattctagtagggctctgcttggtgccaacctcccccaaatgcgcgctgcgggagtgctcttccccaactcaccctagtatcctctcatgtgtgtgcttggtcagcatatctgagacgatgttccgctgtcccagaccagtccagtaatggacgggccagtgtgcgtagtcgtcttccggcttgtccggcgcatgtttggtgaaccggtggggtaaggttggtgtgcccaacgcccgtactcaggggatacctcaaggcacccaggaatgccagggaggtaccccgcttcacagcgggatctgaccctggggtaaatgtctgcgggggggtcttcttggcccacttctcagtacttttcagg  17 Salivirusacatggggggtctgcggacggcttcggcccacccgcgacaagaatgccgtcatctgtcctcatta FHBcccgtattccttcccttcccccgcaaccaccacgcttactcgcgcacgtgttgagtggcacgtgcgttgtccaaacagctacacccacacccttcggggcgggtttgtcccgccctcgggttcctcgcggaacccccccctccctctctctctttctatccgccctcacttcccataactacagtgctttggtaggtgagcaccctgaccccccgcggaagctgctaacgtggcaactgtggggatccaggcaggttatcaaaggcacccggtctttccgccttcaggagtatctctgccggtgaattccggtagggctctgcttggtgccaacctcccccaaatgcgcgctgcgggagtgctcttccccaactcatcttagtaacctctcatgtgtgtgcttggtcagcatatctgaggcgacgttccgctgtcccagaccagtccagcaatggacgggccagtgtgcgtagtcgctttccggttttccggcgcatgtttggcgaaacgctgaggtaaggttggtgtgcccaacgcccgtaatttggtgatacctcaagaccacccaggaatgccagggaggtaccccacttcggtgggatctgaccctgggctaattgtctacggtggttcttcttgcttccacttctcttttttctggcatg  18Salivirustatggcaggcgggcttgtggacggcttcggcccacccacagcaagaatgccatcatctgtcctca NG-J1cccccaattttcccttttcttcccctgcaaccattacgcttactcgcatgtgcattgagtggtgcatgtgttgaacaaacagctacactcacatgggggcgggttttcccgccctacggcctctcgcgaggcccaccccttccctccccttataactacagtgctttggtaggtaagcatcctgatcccccgcggaagctgctcacgtggcaactgtggggacccagacaggttatcaaaggcacccggtctttccgccttcaggagtatccctactagtgaattctagcggggctctgcttggtgccaacctcccccaaatgcgcgctgcgggagtgctcttccccaactcaccctagtatcctctcatgtgtgtgcttggtcagcatatctgagacgatgttccgctgtcccagaccagtccagtaatggacgggccagtgcgtgtagtcgtcttccggcttgtccggggcatgtttggtgaaccggtggggtaaggttggtgtgcccaacgcccgtactttggtgacacctcaagaccacccaggaatgccagggaggtaccccacctcacggtgggatctgaccctgggctaattgtctacggtggttcttcttgcttccacttctttcttctgttcacg  19 HumantttgaaaggggtctcctagagagcttggccgtcgggccttataccccgacttgctgagtttctctaggParecho-agagcccttttcccagccctgaggcggctggtcaataaaagcctcaaacgtaactaacacctaagavirus 1agatcatgtaaaccctatgcctggtctccactattcgaaggcaacttgcaataagaagagtgggatcaagacgcttaaagcatagagacagttttcttttctaacccacatttgtgtggggtggcagatggcgtgccataactctaatagtgagataccacgcttgtggaccttatgctcacacagccatcctctagtaagtttgtgagacgtctggtgacgtgtgggaacttattggaaacaacattttgctgcaaagcatcctactgccagcggaaaaacacctggtaacaggtgcctctggggccaaaagccaaggtttaacagaccctttaggattggttctaaacctgagatgttgtggaagatatttagtacctgctgatctggtagttatgcaaacactagttgtaaggcccatgaaggatgcccagaaggtacccgtaggtaacaagtgacactatggatctgatttggggccagatacctctatcttggtgatctggttaaaaaacatctaatgggccaaacccgggggggatccccggtttcctcttattctatcaatgccact  20 Crohivirusgtataagagacaggtgtttgccttgtcttcggactggcatcttgggaccaaccccccttttccccagc Bcatgggttaaatggcaataaaggacgtaacaactttgtaaccattaagctttgtaattttgtaaccactaagctttgtgcacataatgtaaccatcaagcttgttagtcccagcaggaggtttgcatgcttgtagccgaaatggggctcgaccccccatagtaggatacttgattttgcattccattgtggacctgcaaactctacacatagaggctttgtcttgcatctaaacacctgagtacagtgtgtacctagaccctatagtacgggaggaccgtttgtttcctcaataaccctacataataggctaggtgggcatgcccaatttgcaagatcccagactgggggtcggtctgggcagggttagatccctgttagctactgcctgatagggtggtgctcaaccatgtgtagtttaaattgagctgttcatatacc  21 Yc-3actgaagatcctacagtaactactgccccaatgaacgccacagatgggtctgctgatgactacctatcttagtgctagttgaggtttgaagtgagccggtttttagaagaaccagtttctgaacattatcatccccagcatctattctatacgcacaagatagatagtcatcagcagacacatctgtgctactgcttgatagagttgcggctggtcaacttagattggtataaccagttgagtggcaa  22 Rosavirustatgcatcactggacggcctaacctcggtcgtggcttcttgccgatttcagcgctaccaggctttctg M-7gtctcgccaggcgttgattagtaggtgcactgtctaagtgaagacagcagtgctctctgtgaaaagttgatgacactcttcaggtttgtagcgatcactcaaggctagcggatttccccgtgtggtaacacacgcctctaggcccagaaggcacggtgttgacagcaccccttgagtggctggtcttccccaccagcacctgatttgtggattcttcctagtaacggacaagcatggctgctcttaagcattcagtgcgtccggggctgaaggatgcccagaaggtacccgcaggtaacgataagctcactgtggatctgatctggggctgcgggctgggtgtctttccacccagccaaaacccgtaaaacggtagtcgcagttaaaaaacgtctaggccccacccccccagggatggggggttcccttaaaccctcacaagttcaac  23 Shanbavirustgaaaagggggcgcagggtggtggtggttactaaatacccaccatcgccctgcacttcccttttcc Acctgtggctcagggtcacttagccccctctttgggttaccagtagttttctacccctgggcacagggttaactatgcaagacggaacaacaatctcttagtccccctcgccgatagtgggctcgacccccatgtgtaggagtggataagggacggagtgagccgatacggggaagagtgtgcggtcacaccttaattccatgagcgctgcgaagaaggaagctgtgaacaatggcgacctgaaccgtacacatggagctccacaggcatggtactcgttagactacgcagcctggttgggagtgggtataccctgggtgagccgccagtgaatgggttgttcactggttaacacacactgcctgatagggtcagggcctcctgtccccgccgtaatgaggtagaccatatgcc  24 Pasivirusgcggctggatattctggccgtgcaactgcttttgaccagtggctctgggtaacttagccaaagtgtc Acttctccctttccctattatatgttttatggctttgtctggtcttgtttagtttatatataagatcctttccgccgatatagacctcgacagtctagtgtaggaggattggtgatattaatttgccccagaagagtgaccgtgacacatagaaaccatgagtacatgtgtatccgtggaggatcgcccgggactggattccatatcccattgccatcccaacaagcggagggtatacccactatgtgcacgtctgcagtgggagtctgcagatttagtcatactgcctgatagggtgtgggcctgcactctggggtactcaggctgtttatataat  25Pasivirusgctggactttctggctgcgcaactgcttttaaccagtggctctgggttacttagccaaaaccccctttc A2cccgtaccctagtttgtgtgtgtattattattttgttgttgttttgtaaatttttatataagatcctttccgccgatatagacctcgacagtctagtgtaggaggattggtgatattaatatgccccagaagagtgaccgtgacacatagaaaccatgagtacatgtgtatccgtggaggatcgcccgggactggattccatatcccattgccatcccaacaaacggagggtatacccgctatgtgcgcgtctacagtgggaatctgtagatttagtcatactgcctgatagggtgtgggcctgcactctggggtactcaggctgtttatataat  26Echovirusttaaaacagcctgtgggttgttcccatccacagggcccactgggcgccagcactctggtattgcgg E14taccttagtgcgcctgttttatatacccgtcccccaaacgtaacttagacgcatgtcaacgaagaccaatagtaagcgcagcacaccagctgtgttccggtcaagcacttctgttaccccggaccgagtatcaataagctactcacgtggctgaaggagaaaacgttcgttacccgaccaattacttcaagaaacctagtaacaccatgaaggttgcgcagtgtttcgctccgcacaaccccagtgtagatcaggtcgatgagtcaccgcattccccacgggtgaccgtggcggtggctgcgctggcggcctgcccatggggaaacccatgggacgcttcaatactgacatggtgcgaagagtctattgagctaattggtagtcctccggcccctgaatgcggctaatcctaactgcggagcagatacccacacaccagtgggcagtctgtcgtaacgggcaactctgcagcggaaccgactactttgggtgtccgtgtttctctttatccttatactggctgcttatggtgacaattgagagattgttaccatatagctattggattggccatccggtgacaaatagagcaattgtgtatttgtttgttggtttcgtgccattaaattacaaggttctaaacacccttaatcttattatagcattcaacacaacaaa  27 HumangtacattagatgcgtcatctgcaactttagtcaataaattacctccaatgtcattaccaacattccctacParecho-cttttcactaacacctaagacaacaagtacctatgcctggtctccactattcgaaggcaacttgcaatvirus 5aagaagagtggaattaagacgcttaaagcatagagctagttatcttttctaacccacaaagttttgtggggtggcagatggcgtgccataactctattagtgagataccatgcttgtggatcttatgctcacacagccatcctctagtaagttgataaggtgtctggtgatatgtgggaactcacatgaaccattaatttaccgtaaggtatcctatagccagcggaatcacatctggtgacagatgcctctggggccgaaagccaaggtttaacagaccctataggattggtttcaaaacctgaattgatgtggattgtgtatagtacctgttgatctggtaacagtgtcaacactagttgtaaggcccacgaaggatgcccagaaggtacccgtaggtaacaagtgacactatggatctgatctggggccagctacctctatcatggtgagttggttaaaaaacgtctagtgggccaaacccaggggggatccctggtttccttttacctaatcaaagccact  28 Aichitttgaaaagggggtgggggggcctcggccccctcaccctcttttccggtggtctggtcccggacc Virusaccgttactccattcagcttcttcggaacctgttcggaggaattaaacgggcacccatactccccccaccccccttttgtaactaagtatgtgtgctcgtgatcttgactcccacggaacggaccgatccgttggtgaacaaacagctaggtccacatcctcccttcccctgggagggcccccgccctcccacatcctccccccagcctgacgtatcacaggctgtgtgaagcccccgcgaaagctgctcacgtggcaattgtgggtccccccttcatcaagacaccaggtctttcctccttaaggctagccccggcgtgtgaattcacgttgggcaactagtggtgtcactgtgcgctcccaatctcggccgcggagtgctgttccccaagccaaacccctggcccttcactatgtgcctggcaagcatatctgagaaggtgttccgctgtggctgccaacctggtgacaggtgccccagtgtgcgtaaccttcttccgtctccggacggtagtgattggttaagatttggtgtaaggttcatgtgccaacgccctgtgcgggatgaaacctctactgccctaggaatgccaggcaggtaccccacctccgggtgggatctgagcctgggctaattgtctacgggtagtttcatttccaatccttttatgtcggagtc  29 HepatitisttcaagaggggtctccggagttttccggaacccctcttggaagtccatggtgaggggacttgatacA VirusctcaccgccgtttgcctaggctataggctaaatttccctttccctgtccttcccctatttccctttgtttHA16tgtttgtaaatattaattcctgcaggttcagggttctttaatctgtttctctataagaacactcaatttttcacgctttctgtctcctttcttccagggctctccccttgccctaggctctggccgttgcgcccggcggggtcaactccatgattagcatggagctgtaggagtctaaattggggacgcagatgtttgggacgtcgccttgcagtgttaacttggctttcatgaacctctttgatcttccacaaggggtaggctacgggtgaaacctcttaggctaatacttcaatgaagagatgccttggatagggtaacagcggcggatattggtgagttgttaagacaaaaaccattcaacgccggaggactggctctcatccagtggtgcattgagggattgattgtcagggctgtctctaggtttaatctcagacctctctgtgcttagggcaaacactatttggccttaaatgggatcctgtgagagggggtccctccattgacagctggactgttctttggggccttatgtggtgtttgcctctgaggtactcaggggcatttaggtttttcctcattcttaaataata  30 Phopivirusgggagtaaacctcaccaccgtttgccgtggtttacggctacctatttttggatgtaaatattaattcctgcaggttcaggtctcttgaattatgtccacgctagtggcactctcttacccataagtgacgccttagcggaacctttctacacttgatgtggttaggggttacattatttccctgggccttctttggccctttttcccctgcactatcattctttcttccgggctctcagcatgccaatgttccgaccggtgcgcccgccggggttaactccatggttagcatggagctgtaggccctaaaagtgctgacactggaactggactattgaagcatacactgttaactgaaacatgtaactccaatcgatcttctacaaggggtaggctacgggtgaaaccccttaggttaatactcatattgagagatacttctgataggttaaggttgctggataatggtgagtttaacgacaaaaaccattcaacagctgtgggccaacctcatcaggtagatgcttttggagccaagtgcgtaggggtgtgtgtggaaatgcttcagtggaaggtgccctcccgaaaggtcgtaggggtaatcaggggcagttaggtttccacaattacaatttgaa  31 CVA10gctcttccgatctgggttgttcccacccacagggcccactgggcgccagcactctgattccacggaatctttgtgcgcctgttttacaacccttcccaatttgtacgtagaagcaatacacactactgatcaatagtaggcatggcgcgccagtcatgtcatgatcaagcacttctgttcccccggactgagtatcaatagactgctcacgcggttgaaggagaaaacgttcgttacccggctaactacttcgagaaacctagtagcaccatggaagctgcggagtgtttcgctcagcactttccccgtgtagatcaggtcgatgagtcactgcaatccccacgggcgaccgtggcagtggctgcgttggcggcctgcctatggggcaacccataggacgctaatcctaactgcggagcacatgccttcaacccaggaggtggtgtgtcgtaacgggtaactctggctaatcctaactgcggagcacatgccttcaacccaggaggtggtgtgtcgtaacgggtaactctgcagcggaaccgactactttgggtgtccgtgtttccttttatccttatattggctgcttatggtgacaatcacggaattgttgccatatagctattggattggccatccggtgtctaacagagctattgtatacctatttgttggatttactcccctatcatacaaatctctgaacactttgtgctttatactgaacttaaacacacgaaa 32 Entero-ttaaaacagctctggggttgttcccaccccagaggcccacgtggcggccagtacaccggtaccacvirus Cggtacccttgtacgcctgttttatactccccctccccgtaaactagaagcacgaaacacaagttcaatagaagggggtacagaccagtaccaccacgaacaagcacttctgttcccccggtgaggtcacatagactgtccccacggtcaaaagtgactgatccgttatccgctcacgtacttcggaaagcctagtaccaccttggaatctacgatgcgttgcgctcagcactcgaccccggagtgtagcttaggctgatgagtctggacgttccccactggtgacagtggtccaggctgcgttggcggcctacctgtggtccaaaaccacaggacgctagtagtgaacaaggtgtgaagagcccactgagctacctgagaatcctccggcccctgaatgcggctaatcccaaccacggagcaggtaatcgcaaaccagcggtcagcctgtcgtaacgcgtaagtctgtggcggaaccgactactttgggtgtccgtgtttccttttatttttatggtggctgcttatggtgacaatcatagattgttatcataaagcaaattggattggccatccggagtgagctaaactatctatttctctgagtgttggattcgtttcacccacattctgaacaatcagcctcattagtgttaccctgttaataagacgatatcatcacg  33 Entero-ttaaaacagctctggggttgttcccaccccagaggcccacgtggcggctagtactccggtaccccvirus Dggtacccttgtacgcctgttttatactccctttcccaagtaactttagaagaaataaactaatgttcaacaggagggggtacaaaccagtaccaccacgaacacacacttctgtttccccggtgaagttgcatagactgtacccacggttgaaagcgatgaatccgttacccgcttaggtacttcgagaagcctagtatcatcttggaatcttcgatgcgttgcgatcagcactctaccccgagtgtagcttgggtcgtgagtctggacaccccacaccggcgacgtggtccaggctgcgttggcggcctaccctggctagcaccatgggacgctagttgtgaacaaggtgcgaagagcctattgagctacctgagagtcctccggcccctgaatgcggctaatcccaaccacggagcaaatgctcacaatccagtgagtggtttgtcgtaatgcgcaagtctgtggcggaaccgactactttgggtgtccgtgtttccttttatttttattatggctgcttatggtgacaatctgagattgttatcatatagctattggattagccatccggtgatatcttgaaattttgccataactttttcacaaatcctacaacattacactacactttctcttgaataattgagacaactcata  34 Entero-ttaaaatagcctcagggttgttcccaccctgagggcccacgtggtgtagtactctggtattacggtacvirus Jctttgtacgcctattttatacccccttccccaagtaatttagaagcaagcacaaaccagttcagtagtaagcagtacaatccagtactgtaatgaacaagtacttctgttaccccggaagggtctatcggtaagctgtacccacggctgaagaatgacctaccgttaaccggctacctacttcgagaagcctagtaatgccgttgaagttttattgacgttacgctcagcacactaccccgtgtgtagttttggctgatgagtcacggcactccccacgggcgaccgtggccgtggctgcgttggcggccaaccaaggagtgcaagctccttggacgtcatattacagacatggtgtgaagagcctattgagctaggtggtagtcctccggcccctgaatgcggctaatcctaactccggagcatatcggtgcgaaccagcacttggtgtgttgtaatacgtaagtctggagcggaaccgactactttgggtgtccgtgtttcctgttttaacttttatggctgcttatggtgacaatttaacattgttaccatatagctgagggttggccatccggattttgttataaaaccatttcctcgtgccttgacctttaacacatttgtgaacttctttaaatcccttttattagtccttaaatactaaga  35 HumanaactgttgttgtagcaatgcgcatattgctacttcggtacgcctaattggtaggcgcccggccgaccPegivirusggccccgcaagggcctagtaggacgtgtgacaatgccatgagggatcatgacactggggtgag 2cggaggcagcaccgaagtcgggtgaactcgactcccagtgcgaccacctggcttggtcgttcatggagggcatgcccacgggaacgctgatcgtgcaaagggatgggtccctgcactggtgccatgcgcggcaccactccgtacagcctgatagggtggcggcgggcccccccagtgtgacgtccgtggag cgcaac 36 GBV-C tgacgtgggggggttgatTTTccccccccggcactgggtgcaagccccagaaaccgacgcctGT110 atctaagtagacgcaatgactcggcgccgactcggcgaccggccaaaaggtggtggatgggtgatgacagggttggtaggtcgtaaatcccggtcatcctggtagccactataggtgggtcttaagagaaggtcaagattcctcttacgcctgcggcgagaccgcgcacggtccacaggtgttggccctaccggtgtgaataagggcccgacatcaggc  37 GBV-CgacgtgggggggttgatccccccccTTTggcactgggtgcaagccccagaaaccgacgccta K1737tttaaacagacgttaagaaccggcgccgacccggcgaccggccaaaaggtggtggatgggtgatgccagggttggtaggtcgtaaatcccggtcatcttggtagccactataggtgggtcttaagggttggttaaggtccctctggcgcttgtggcgagaaagcgcacggtccacaggtgttgccctaccggtgtgaataagggcccgacgtcaggctcgtcgttaaaccgagcccactacccacctgggcaaacaacgcccacgtacggtccacgtcgcccttcaatgtctctcttgaccaataggcttagccggcgagttgacaaggaccagtgggggctgggcggtaggggaaggacccctgccgctgcccttcccggtggagtg ggaaatgc 38 GBV-CtgacgtgggggggttgatccGccccccccggcactCggtgcaagccccataaaccgacgccta Iowatctaagtagacgcaatgactcggcgccgactcggcgaccggccaaaaggtggtggatgggtggtgacagggttggtaggtcgtaaatcccggtcatcctggtagccactataggtgggtcttaagagaaggtcaagactcctcttgtgcctgcggcgagaccgcgcacggtccacaggtgctggccctaccggtgtgaataagggcccgacgtcaggctcgtcgttaaaccgagcccgtcacccacctgggcaaacgacgcccacgtacggtccacgtcgcccttca  39 PegivirustgtagcaatgcgcatattgctacttcggtacgcctaattggtaggcgcccggccgaccggccccgcA 1220 aagggcctagtaggacgtgtgacaatgccatgcgggatcatgacactggggtgagcggaggcagcaccgaagtcgggtgaactcgactcccagtgcgaccacctggcaggtcgttcatggagggcatgcccacgggaacgctgatcgtgcaaagggatgggtccctgcactggtgccatgcgcggcaccactccgtacagcctgatagggtggcggcgggcccccccagtgtgacgtccgtggagcgcaac  40PasivirusattttctggccgtgtagctgcttttgaccagtggctctgggttacttagccaaatcccccttccttcaccA3cttttaaatttgatggtctgtgttgtttgttttgtcttgtctaaataatatataagatccttcccgccgatacagacctcgacagtctggtgtaggagggttggtgttattaatttgccccagaagagtgaccgtgacacatagaaaccatgagtacatgtgtatccgtggaggatcgcccgggactggattccatatcccattgccatcccaacaagcggagggtatacccactatgtgcgcgtttgcagtgggaatctgcaaatttagtcatactgcctgatagggtgtgggcctgcactctggggtactcaggctgacatataat  41 Sapeloviruscccctccacccttaaggtggttgtatcccacataccccaccctcccttccaaagtggacggacaactggattttgactaacggcaagtctgaatggtatgatttggatacgtttaaacggcagtagcgtggcgagctatggaaaaatcgcaattgtcgatagccatgttagtgacgcgcttcggcgtgctcctttggtgattcggcgactggttacaggagagtaggcagtgagctatgggcaaacctctacagtattacttagagggaatgtgcaattgagacttgacgagcgtctctttgagatgtggcgcatgctcttggcattaccatagtgagcttccaggagggaaacctggactgggcctatactacctgatagggtcgcggctggccgcctgtaactagtatagtcagttgaaaccccccc  42 Rosavirusgtctctttagtgtctatgcttcagagagcggtgaactgacaccgttgcttcttgcacagcccttcgtgc Bcggtctttccggttctcgacagcgttgggcatcatggctagttaggctaagatagtggatgatctagtgaacagttttggattgtttggagttttgtagcgatgctagtagtgtgtgtggacctccccacgtggtaacacgtgccccacaggccaaaagccaaggtgttgaaagcacccctactagtcccagactcacccatctgggaactcctctcatgaaaaatcttagtaacttttgattcggctattcatcaacctctctagtcaagggctgaaggatgcccggaaggtacccgcaggtaacgataagctcactgtggatctgatccggggctttggtgcgaccgtctgtccggcgtagccagagttaaaaaacgtctaggcccttccaccccaagggattggggtttccccaatcatttgaaagttcact  43 BakunsattttgaacgccacctcggagcgatatccggggaccccctcccctttttccttcctaccttcttcccaaaVirustttccctcttcccttgttattttggtttggatttcctggacatgactcggacggatctatctcatttgctttgtgtctgctccaccagtggcatggtcgaaagatcatcaacactggacgtgtactgtaatggccaaacgtgcccacaggggaaaccatgccggtcgctgtagcggcgggtggacgtggtggacccctctccctgctcataaactttgggtaggtgaagggttcaagcgacgcttgccgtgagggcgcatccggatggtgggaaccaacaaactaggctgtaatggccgacctcaggtggatgagctagggctgctgcaccaaaagggactcgattcgatatcccggcctggtagcctagtgcagtggactcgtagttgggaatctacgactggcctagtacagggtgatagccccgtttcccacgcccacctgttgtagggacacccccccc  44Tremovirustttgaaagaggcctccggagtgtccggaggctctctttcgacccaacccatactggggggtgtgtg Aggaccgtacctggagtgcacggtatatatgcattcccgcatggcaagggcgtgctaccttgccccttgacgcatggtatgcgtcatcatttgccttggttaagccccatagaaacgaggcgtcacgtgccgaaaatccctttgcgtttcacagaaccatcctaaccatgggtgtagtatgggaatcgtgtatggggatgattaggatctctcgtagagggataggtgtgccattcaaatccagggagtactctggctctgacattgggacatttgatgtaaccggacctggttcagtatccgggttgtcctgtattgttacggtgtatccgtcttggcacactgaaagggtatttttgggtaatcctttcctactgcctgatagggtggcgtgcccggccacgagagattaagggtagcaatttaaac  45 SwinegcttttgaccagtggctctgggttacttagccaagtccctttctcttattttcactagtttatgttgtgtPasivirusgttgtctgttttgttttgtttaaattgtatacaagatccttcccgccgacacagacctcgacagtctggt 1gtaggagggttggtgatattaatttgccccaaaagagtgaccgtgatacgtggaaaccatgagtacatgtgtatccgtggaggatcgcccgggactggattccatatcccattgccatcccaacaaacggagggtatacccaccacgtgcgcgtttgcagtgggaatctgcaaatttagtcatactgcctgatagggtgtgggcctgcactttggggtactcaggctgttcatataat  46 PLV-CHNacatggggtatgttgtctgtcctgttttgagaaacaatatataagatcctttccgccgatatagacctcgacagtctagtgtaggaggattggtgatagtaacttgccccagaagagtgaccgtgacacatagaaaccatgagtacatgtgtatccgtggaggatcgcccgggactggattccatatcccattgccatcccaacaaacggagggtatacccactatgtgcgcgtttgcagtgggagcctgcaaatttagtcatactgcctgatagggtgtgggcctgcactctggggtactcaggctgtttatataat  47 Pasivirustgaaaaagtggttgtgcagctggattttccggctgtgcaactgcttttgaccagtggctctgggttact Atagccaaattcctttcccttatccctattggtttgtgttgtgtgttgtttgttttgttttgtcttaacta(longer)tatacaagatccttcccgccgatacagacctcgacagtctggtgtaggagggttggtgttattaatttgccccaaaagagtgaccgtgacacgtggaaaccatgagtacatgtgtatccgtggaggatcgcccgggactggattccatatcccattgccatcccaacaaacggagggtatacccaccacgtgcgcgtttgcagtgggaatctgcaaatttagtcatactgcctgatagggtgtgggcctgcactttggggtactcaggctgtttatataat  48 Sicinivirusgtgtcattaaggtgtgtttggaagttcgaattagctggtttgtggtgattagtagaccccctggaggtacccaattcggatctgaccagggacccgtgactataccgctccggtaattcgggtttaaaacaatgaacgtcaccacacaattacttttctcattttattttcatcattgtcttcctatttaccgattacactcgatttccttggatgttcctggagatttccctggttacctggaccctcattattgttgttgatcacccagcgagctgtcccaattgcttattatttgcgcttacaacacgtcctaatatttttctggagatcgggagattgagctcccgggctatcctgccattcaac  49 Hepacivirusgggaacaatggtccgtccgcggaacgactctagccatgagtctagtacgagtgcgtgccacccat Ktagcacaaaaaccactgactgagccacacccctcccggaatcctgagtacaggacattcgctcggacgacgcatgagcctccatgccgagaaaattgggtatacccacgggtaaggggtggccacccagcgggaatctgggggctggtcactgactatggtacagcctgatagggtgctgccgcagcgtcagtggtatgcggctgttcatggaac  50 Hepacivirusacctccgtgctaggcacggtgcgttgtcagcgttttgcgcttgcatgcgctacacgcgtcgtccaac Agcggagggaacttcacatcaccatgtgtcactccccctatggagggttccaccccgcttacacggaaatgggttaaccatacccaaagtacgggtatgcgggtcctcctagggcccccccggcaggtcgagggagctggaattcgtgaattcgtgagtacacgaaaatcgcggcttgaacgtctttgaccttcggagccgaaatttgggcgtgccccacgaaggaaggcgggggcggtgttgggccgccgccccccttatcccacggtctgataggatgcttgcgagggcacctgccggtctcgtagaccataggac  51 BVDV1gtatacgagaatttgcctaggacctcgtttacaatatgggcaatctaaaattataattaggcctaagggacaaatcctcctcagcgaaggccgaaaagaggctagccatgcccttagtaggactagcaaaataaggggggtagcaacagtggtgagttcgttggatggctgaagccctgagtacagggtagtcgtcagtggttcgacgcttcggaggacaagcctcgagataccacgtggacgagggcatgcccacagcacatcttaacctggacgggggtcgttcaggtgaaaacggtttaaccaaccgctacgaatacagcctgatagggtgctgcagaggcccactgtattgctactgaaaatctctgctgtacatggcac  52 BordergtatacgggagtagctcatgcccgtatacaaaattggatattccaaaactcgattgggttagggagcDisease cctcctagcgacggccgaaccgtgttaaccatacacgtagtaggactagcagacgggaggactaVirus gccatcgtggtgagatccctgagcagtctaaatcctgagtacaggatagtcgtcagtagttcaacgcaggcacggttctgccttctagatgctacgtggacgagggcatgcccaagacttgctttaatctcggcgggggtcgccgaggtgaaaacacctaacggtgttggggttacagcctgatagggtgctgcagaggcccacgaataggctagtataaaaatctctgctgtacatggcac  53 BVDV2gtatacgagattagctaaagtactcgtatatggattggacgtcaacaaatttttaattggcaacgtagggaaccttcccctcagcgaaggccgaaaagaggctagccatgccccttagtaggactagcaaaagtagggggactagcggtagcagtgagttcgttggatggccgaacccctgagtacaggggagtcgtcaatggttcgacactccattagtcgaggagtctcgagatgccatgtggacgagggcatgcccacggcacatcttaacccatgcgggggttgcatgggtgaaagcgctaatcgtggcgttatggacacagcctgatagggtgtagcagagacctgctattccgctagtaaaaaactctgctgtacatggcac  54 CSFV-gtatacgaggttagttcattctcgtatgcattattggacaaatcaaaatttcaatttggttcagggcctcPK15C cctccagcgacggccgaactgggctagccatgcccatagtaggactagcaaacggagggactagccgtagtggcgagctccctgggtgttctaagtcctgagtacaggacagtcgtcagtagttcgacgtgagcagaagcccacctcgagatgctatgtggacgagggcatgcccaagacgcaccttaaccctagcgggggtcgctagggtgaaatcacaccacgtgatgggagtccgacctgatagggtgctgcagaggctcactattaggctagtataaaaatctctgctgtacatggcac  55 SF573aaaaccgaccccagagatcagaaagtcgttgacgcgatcttttattagaggacgttgcgctggcgcDicistro-gagctttaattagcagacgccaaaaataaacaacaaaatgctgatcgcgagacttaattgtcagac virusgattggccaaatccgatgtgatctttgctgctcccagattgccgaaataggagtagtag  56 HubeiccccaaaaccccccccttaaactcaacactgtagtggattcattttccgttgcaaaacaaaacattacPicorna-tacccgcatttatgtaggctctgtgttttctatgcgaccgttacattaatctctactctgacccactagtlike Virusttataaaaccgaagacctgaatgaaacgattttccttcttttcaacctctaacgaacctctgacggcttgagaaacctgaagttagtaattatgtttaaaagaaaggaaagtcaaacgcgatgactcttacatccctattccataccgttgctccacaatgtgagcgatgcgaggtcgggactgcagtattaggggaacgagctacatggagagttaattatctctcccctcctacgggagtctcatgtgagctgtagaaagcggttggcacctctcgttacctcgcctgtacatgatcc  57 CRPVaaaagcaaaaatgtgatcttgcttgtaaatacaattttgagaggttaataaattacaagtagtgctatttttgtatttaggttagctatttagctttacgttccaggatgcctagtggcagccccacaatatccaggaagccctctctgcggtttttcagattaggtagtcgaaaaacctaagaaatttacct  58 Salivirustttcctcctttcgaccgccttacggcaggcgggtccgcggacggcttcggcctacccgcgacaag A BN5aatgccgtcatctgtccttatcacccatattctttcccttcccccgcaaccatcacgcttactcgcgcacgtgttgagtggcacgtgcgttgtccaaacagttacactcacacccttggggcgggtttgtcccgccctcgggttcctcgcggaaccctccctcttctctctccctttctatccgccttcactttccataactacagtgctttggtaggtaagcatcctgaccccccgcggaagctgccaacgtggcaactgtggggatccaggcaggttatcaaaggcacccggtctttccgccttcaggagtatccctgccggtgaattccgacagggctctgcttggtgccaacctcccccaaatgcgcgctgcgggagtgctcttccccaactcatcttagtaacctctcatgtgtgtgcttggtcagcatatctgaggcgacgttccgctgtcccagaccagtccagcaatggacgggccagtgtgcgtagtcgctttccggtttcccggcgcatgtttggcgaaacgctgaggtaaggttggtgtgcccaatgcccgtaatttggtgacacctcaagaccacccaggaatgccagggaggtaccccacttcggtgggatctgaccctgggctaattgtctacggtggttcttcttgcttccacttctcttttttctggcatg  59 Salivirustatggcaggcgggcttgtggacggcttcggcccacccacagcaagaatgccatcatctgtcctca A BN2cccccatgtttcccctttctttccctgcaaccgttacgcttactcgcaggtgcatttgagtggtgcacgtgttgaataaacagctacactcacatgggggcgggttttcccgccctgcggcctctcgcgaggcccacccctccccttcctcccataactacagtgctttggtaggtaagcatcctgatcccccgcggaagctgctcacgtggcaactgtggggacccagacaggttatcaaaggcacccggtctttccgccttcaggagtatccctgctagtgaattctagtagggctctgcttggtgccaacctcccccaaatgcgcgctgcgggagtgctcttccccaactcaccctagtatcctctcatgtgtgtgcttggtcagcatatctgagacgatgttccgctgtcccagaccagtccagtaatggacgggccagtgtgcgtagtcgtcttccggcttttccggcgcatgtttggtgaaccggtggggtaaggttggtgtgcccaacgcccgtactttggtgatacctcaagaccacccaggaatgccagggaggtaccccgcttcacagcgggatctgaccctgggctaattgtctacggtggttcttcttgcttccacttctttctactgttc  60 Salivirustttcgaccgccttatggcaggcgggcttgtggacggcttcggcccacccacagcaagaatgccat Acatctgtcctcacccccatttctcccctccttcccctgcaaccattacgcttactcgcatgtgcattgag02394 tggtgcacgtgttgaacaaacagctacactcacgtgggggcgggttttcccgcccttcggcctctcgcgaggcccacccttccccttcctcccataactacagtgctttggtaggtaagcatcctgatcccccgcggaagctgctcgcgtggcaactgtggggacccagacaggttatcaaaggcacccggtctttccgcctccaggagtatccctgctagtgaattctagtggggctctgcttggtgccaacctcccccaaatgcgcgctgcgggagtgctcttccccaactcaccctagtatcctctcatgtgtgtgcttggtcagcatatctgagacgatgttccgctgtcccagaccagtccagcaatggacgggccagtgtgcgtagtcgtcttccggcttgtccggcgcatgtttggtgaaccggtggggtaaggttggtgtgcccaacgcccgtactttggtgacaactcaagaccacccaggaatgccagggaggtaccccgcctcacggcgggatctgaccctgggctaattgtctacggtggttcttcttgcttccatttctttcttctgttc  61 Salivirustatggcaggcgggcttgtggacggtttcggcccacccacagcaagaatgccatcatctgtcctcac A GUTccccaattttccctttcttcccctgcaatcatcacgcttactcgcatgtgcattgagtggtgcatgtgttgaacaaacagctacactcacatgggggcgggttttcccgccctacggcctctcgcgaggcccacccttcccctccccttataactacagtgctttggcaggtaagcatcctgatcccccgcggaagctgctcacgtggcaactgtggggacccagacaggttatcaaaggcacccggtctttccgccttcaggagcatccccactagtgaattctagtggggctctgcttggtgccaacctcccccaaatgcgcgctgcgggagtgctcttccccaacccatcctagtatcctctcatgtgtgtgcttggtcagcatatctgagacgacgttccgctgtcccagaccagtccagtaatggacgggccagtgtgcgtagtcgtcttccggcttgtccggcgcatgtttggtgaaccggtggggtaaggaggtgtgcccaacgcccgtactttggtgacacctcaagaccacccaggaatgccagggaggtaccccgcctcacggcgggatctgaccctgggctaattgtctacggtggttcttcttgcttccacttctttctt  62 SalivirusttctcctgcaaccattacgcttaatcgcatgtgcattgagtggtgcatgtgttgaacaaacagctacaA CH atcacatgggggcgggttttcccgccccacggcttctcgcgaggcccatccctcccttttctcccataactacagtgctttggtaggtaagcatcccgatctcccgcggaagctgctcacgtggcaactgtggggacccagacaggttatcaaaggcacccggtctttccgccttcaggagtatccctgctagcgaattctagtagggctctgcttggtgccaacctctcccaaatgcgcgctgcgggagtgctcttccccaaatcaccccagtatcctctcatgtgtgtgcctggtcagcatatctgagacgatgttccgctgtcccagaccagtccagtaatggacgggccagtgtgcgtagtcgtcctccggcttgtccggcgcatgtttggtgaaccggtggggtaaggttggtgtgcccaacgcccgtaatcaggggatacctcaaggcacccaggaatgccagggaggtatcccgcctcacagcgggatctgaccctggggtaaatgtctgcggggggtcctcttggcccaattctcagtaattttcagg  63 SalivirustctgtcctcaccccatcttcccttctttcctgcaccgttacgcttactcgcatgtgcattgagtggtgcaA SZ1 cgtgcttgaacaaacagctacactcacatgggggcgggttttcccgccctgcggcctctcgcgaggcccacccctccccttcctcccataactacagtgctttggtaggtaagcatcctgatcccccgcggaagctgctcacgtggcaactgtggggacccagacaggttatcaaaggcacccggtctttccgccttcaggagtatccctgctagtgaattctagtagggctctgcttggtgccaacctcccccaaatgcgcgctgcgggagtgctcttccccaactcaccctagtatcctctcatgtgtgtgcttggtcagcatatctgagacgatgttccgctgtcccagaccagtccagtaatggacgggccagtgtgcgtagtcgtcttccggcttgtccggcgcatgtttggtgaaccggtggggtaaggttggtgtgcccaacgcccgtactttggtgatacctcaagaccacccaggaatgccagggaggtaccccgcttcacagcgggatctgaccctgggctaattgtctacggtggttcttcttgcttccacttctttctactgttcatg  64 Salivirusacatggggggtctgcggacggcttcggcccacccgcgacaagaatgccgtcatctgtcctcatta FHBcccgtattccttcccttcccccgcaaccaccacgcttactcgcgcacgtgttgagtggcacgtgcgttgtccaaacagctacacccacacccttcggggcgggtttgtcccgccctcgggttcctcgcggaacccccccctccctctctctctttctatccgccctcacttcccataactacagtgctttggtaggtgagcaccctgaccccccgcggaagctgctaacgtggcaactgtggggatccaggcaggttatcaaaggcacccggtctttccgccttcaggagtatctctgccggtgaattccggtagggctctgcttggtgccaacctcccccaaatgcgcgctgcgggagtgctcttccccaactcatcttagtaacctctcatgtgtgtgcttggtcagcatatctgaggcgacgttccgctgtcccagaccagtccagcaatggacgggccagtgtgcgtagtcgctttccggttttccggcgcatgtttggcgaaacgctgaggtaaggttggtgtgcccaacgcccgtaatttggtgatacctcaagaccacccaggaatgccagggaggtaccccacttcggtgggatctgaccctgggctaattgtctacggtggttcttcttgcttccacttctcttttttctggcatg  65CVB3 ttaaaacagcctgtgggttgatcccacccacaggcccattgggcgctagcactctggtatcacggtacctttgtgcgcctgttttataccccctcccccaactgtaacttagaagtaacacacaccgatcaacagtcagcgtggcacaccagccacgttttgatcaagcacttctgttaccccggactgagtatcaatagactgctcacgcggttgaaggagaaagcgttcgttatccggccaactacttcgaaaaacctagtaacaccgtggaagttgcagagtgtttcgctcagcactaccccagtgtagatcaggtcgatgagtcaccgcattccccacgggcgaccgtggcggtggctgcgttggcggcctgcccatggggaaacccatgggacgctctaatacagacatggtgcgaagagtctattgagctagttggtagtcctccggcccctgaatgcggctaatcctaactgcggagcacacaccctcaagccagagggcagtgtgtcgtaacgggcaactctgcagcggaaccgactactttgggtgtccgtgtttcattttattcctatactggctgcttatggtgacaattgagagatcgttaccatatagctattggattggccatccggtgactaatagagctattatatatccctttgttgggtttataccacttagcagaaagaggttaaaacattacaattcattgttaagttgaatacagcaaa  66 CVB1ttaaaacagcctgtgggttgttcccacccacaggcccattgggcgctagcactctggtatcacggtacctttgtgcgcctgttttacatcccctccccaaattgtaatttagaagtttcacacaccgatcattagcaagcgtggcacaccagccatgttttgatcaagcacttctgttaccccggactgagtatcaatagaccgctaacgcggttgaaggagaaaacgttcgttacccggccaactacttcgaaaaacctagtaacaccatggaagttgcggagtgtttcgctcagcactaccccagtgtagatcaggtcgatgagtcaccgcgttccccacgggcgaccgtggcggtggctgcgttggcggcctgcctacggggaaacccgtaggacgctctaatacagacatggtgcgaagagtctattgagctagttggtaatcctccggcccctgaatgcggctaatcctaactgcggagcacataccctcaaaccagggggcagtgtgtcgtaacgggcaactctgcagcggaaccgactactttgggtgtccgtgtttcattttattcctatactggctgcttatggtgacaattgacaggttgttaccatatagttattggattggccatccggtgactaacagagcaattatatatctctttgttgggtttataccacttagcttgaaagaggttaaaacactacatctcatcattaaactaaatacaacaa a 67 Echovirusttaaaacagcctgtgggttgttcccacccacagggcccattgggcgtcagcaccctggtatcacgg 7tacctttgtgcgcctgttttatatcccttcccccaattgtaacttagaagaaacacacaccgatcaacagcaagcgtggcacaccagccatgttttggtcaagcacttctgttaccccggactgagtatcaatagactgctcacgcggttgaaggagaaagcgtccgttatccggccagctacttcgagaaacctagtaacaccatggaagttgcggagtgtttcgctcagcactaccccagtgtagatcaggtcgatgagtcaccgctttccccacgggcgaccgtggcggtggctgcgttggcggcctgcctatgggggaacccataggacgctctaatacagacatggtgcgaagagtctattgagctagctggtattcctccggcccctgaatgcggctaatcctaactgtggagcacatgcccctaatccaaggggtagtgtgtcgtaatgagcaattccgcagcggaaccgactactttgggtgtccgtgtttcctcttattcttgtactggctgcttatggtgacaattgagagattgttaccatatagctattggattggccatccggtgactaatagagctattgtgtatctctttgttggatttgtaccacttaatttgaaagaaatcaggacactacgctacattttactattgaacaccgca aa 68 CVB5ttaaaacagcctgtgggttgtacccacccacagggcccactgggcgctagcactctggtatcacggtacctttgtgcgcctgttttatgcccccttcccccaattgaaacttagaagttacacacaccgatcaacagcgggcgtggcataccagccgcgtcttgatcaagcactcctgtttccccggaccgagtatcaatagactgctcacgcggttgaaggagaaaacgttcgttacccggctaactacttcgagaaacctagtagcatcatgaaagttgcgaagcgtttcgctcagcacatccccagtgtagatcaggtcgatgagtcaccgcattccccacgggcgaccgtggcggtggctgcgttggcggcctgcctacggggcaacccgtaggacgcttcaatacagacatggtgcgaagagtcgattgagctagttagtagtcctccggcccctgaatccggctaatcctaactgcggagcacataccctcaacccagggggcattgtgtcgtaacgggtaactctgcagcggaaccgactactttgggtgtccgtgtttccttttattcttataatggctgcttatggtgacaattgaaagattgttaccatatagctattggattggccatccggtgtctaacagagctattatatacctctttgttggatttgtaccacttgatctaaaggaagtcaagacactacaattcatcatacaattgaacacagcaaa  69 EVA71ttaaaacagcctgtgggttgcacccactcacagggcccactgggcgcaagcactctggcacttcggtacctttgtgcgcctgttttatatcccctcccccaatgaaatttagaagcagcaaaccccgatcaatagcaggcataacgctccagttatgtcttgatcaagcacttctgtttccccggactgagtatcaatagactgctcacgcggttgaaggagaaaacgttcgttatccggctaactacttcggaaagcctagtaacaccatggaagttgcggagagtttcgttcagcacttccccagtgtagatcaggtcgatgagtcaccgcattccccacgggcgaccgtggcggtggctgcgttggcggcctgcccatggggtaacccatgggacgctctaatacggacatggtgtgaagagtctactgagctagttagtagtcctccggcccctgaatgcggctaatcccaactgcggagcacacgcccacaagccagtgggtagtgtgtcgtaacgggcaactctgcagcggaaccgactactttgggtgtccgtgtttccttttattcttatgttggctgcttatggtgacaattaaagagttgttaccatatagctattggattggccatccggtgtgcaacagagcgatcgtttacctatttattggttttgtaccattgacactgaagtctgtgatcacccttaattttatcttaaccctcaacacagccaaac  70 CVA3ttaaaacagcctgtgggttgtacccacccacagggcccactgggcgctagcacactggtattacggtacctttgtgcgcctgttttataccccccccaacctcgaaacttagaagtaaagcaaacccgatcaatagcaggtgcggcgcaccagtcgcatcttgatcaagcacttctgtaaccccggaccgagtatcaatagactgctcacgcggttgaaggagaaaacgttcgttacccggctaactacttcgagaaacccagtagcatcatgaaagttgcagagtgtttcgctcagcactacccccgtgtagatcaggccgatgagtcaccgcacttccccacgggcgaccgtggcggtggctgcgttggcggcctgcctatggggcaacccataggacgctctaatacggacatggtgcgaagagtctattgagctagttagtagtcctccggcccctgaatgcggctaatcctaactgcggagcacatacccttaatccaaagggcagtgtgtcgtaacgggtaactctgcagcggaaccgactactttgggtgtccgtgtttccttttaatttttactggctgcttatggtgacaattgaggaattgttgccatatagctattggattggccatccggtgactaacagagctattgtgttccaatttgttggatttaccccgctcacactcacagtcgtaagaacccttcattacgtgttatttctcaactcaagaaa  71 CVA12ttaaaacagcctgtgggttgtacccacccacagggcccactgggcgctagcactctggtactacggtacctttgtgtgcctgttttaagcccctaccccccactcgtaacttagaaggcttctcacactcgatcaatagtaggtgtggcacgccagtcacaccgtgatcaagcacttctgttaccccggtctgagtaccaataagctgctaacgcggctgaaggggaaaacgatcgttatccggctaactacttcgagaaacccagtaccaccatgaacgttgcagggtgtttcgctcggcacaaccccagtgtagatcaggtcgatgagtcaccgtattccccacgggcgaccgtggcggtggctgcgttggcggcctgcccatggggtgacccatgggacgctctaatactgacatggtgcgaagagtctattgagctagttagtagtcctccggcccctgaatgcggctaatcctaactgcggagcacatacccttaatccaaagggcagtgtgtcgtaacgggcaactctgcagcggaaccgactactttgggtgtccgtgtttccttttattcttacattggctgcttatggtgacaattgaaaagttgttaccatatagctattggattggccatccggtgacaaatagagctattgtatatctttttgttggttacgtaccccttaattacaaagtggtttcaactttgaaatacatcctaacactaaattgtagaaa  72 EV24ttaaaacagcctgtgggttgcacccacccacagggcccacagggcgctagcactctggtatcacggtacctttgtgcgcctgttttattaccccttccccaattgaaaattagaagcaatgcacaccgatcaacagcaggcgtggcgcaccagtcacgtctcgatcaagcacttctgtttccccggaccgagtatcaatagactgctcacgcggttgaaggagaaagtgttcgttatccggctaaccacttcgagaaacccagtaacaccatgaaagttgcagggtgtttcgctcagcacttccccagtgtagatcaggtcgatgagtcaccgcgttccccacgggcgaccgtggcggtggctgcgttggcggcctgcctatgggttaacccataggacgctctaatacagacatggtgcgaagagtttattgagctggttagtatccctccggcccctgaatgcggctaatcctaactgcggagcacgtgcctccaatccagggggttgcatgtcgtaacgggtaactctgcagcggaaccgactactttgggtgtccgtgtttccttttattcttatactggctgcttatggtgacaatcgaggaattgttaccatatagctattggattggccatccggtgtctaacagagcgattatatacctctttgttggatttatgcagctcaataccaccaactttaacacattgaaatatatcttaaagttaaacacagcaaa 348 AP1.0attctcgggctacggccctggagccactccggctcctaaagatttagaagtttgagcacacccgcccactagggccccccatccaggggggcaacgggcaagcacttctgtttccccggtatgatctgataggctgtaaccacggctgaaacagagattatcgttatccgcttcactacttcgagaagcctagtaatgatgggtgaaattgaatccgttgatccggtgtctcccccacaccagaaactcatgatgagggttgccatcccggctacggcgacgtagcgggcatccctgcgctggcatgaggcctcttaggaggacggatgatatggatcttgtcgtgaagagcctattgagctagtgtcgactcctccgcccccgtgaatgcggctaatcctaaccccggagcaggtgggtccaatccagggcctggcctgtcgtaatgcgtaagtctgggacggaaccgactactttcgggaaggcgtgtttccatttgttcattatttgtgtgtttatggtgacaactctgggtaaacgttctattgcgtttattgagagattcccaacaattgaacaaacgagaactacctgttttattaaatttacacagagaagaattaca 349 CK1.0gtggccacgcccgggccaccgatacttcccttcactccttcgggactgttggggaggaacacaacagggctcccctgttttcccattccttcccccttttcccaaccccaaccgccgtatctggtggcggcaagacacacgggtctttccctctaaagcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcgggagtgctcccacccaactgttgtaagcctgtccaacgcgtcgtcctggcaagactatgacgtcgcatgttccgctgcggatgccgaccgggtaaccggttccccagtgtgtgtagtgcgatcttccaggtcctcctggttggcgttgtccagaaactgcttcaggtaagtggggtgtgcccaatccctacaaaggttgattctttcaccaccttaggaatgctccggaggtaccccagcaacagctgggatctgaccggaggctaattgtctacgggtggtgtttcctttttcttttcacacaactctactgctgacaactcactgactatccacttgctctgTcacG 350 PV1.0aacaaaaggctacaccacttgggctacggcccgcgccaccttgtggcgcaaagacattagaagaatagcataccgcccactagggccctgcagccagcagggtaacgggcaagcacttctgtctccccggtagaacggtataggctgtacccacggccgaaaactgaactatcgttacccgactccgtacttcgcaaagcttagtaggaaactggaaagttcgagttattgacccggagtgttccccccactccagaaacgcgtgatgagggttgccaccccgaccatggcgacatggtgggcatccctgcgctggcacgcggcctctaagaggataactcgctcctactggtaaccgaagagccccgtgagctacggtttattcctccgcctccctgaatgcggctaatcctaacccatgagcagttgccatagatccatatggtggactgtcgtaacgcgtaagttgtgggcggaaccgactactttgggatggcgtgtttccttgttttctccatttgttgttgtatggtgacaagttatagatctcgatctatagcgtttcttgagagatttccaaacatttattcaagtcgtacaattcttgtgtttaagcagtacagtgtaacc 351 SV1.0tctgtcctcaccccatcttcccttctttcctgcaccgttacgcttactcgcatgtgcattgagtggtgcacgtgcttgaacaaacagctacactcacatgggggcgggttttcccgccctgcggcctctcgcgaggcccacccctccccttcctcccataactacagtgctttggtaggtaagcatcctgatcccccgcggaagctgctcacgtggcaactgtggggacccagacaggttatcaaaggcacccggtctttccgccttcaggagtatccctgctagtgaattctagtagggctctgcttggtgccaacctcccccaaatgcgcgctgcgggagtgctcttccccaactcaccctagtatcctctcatgtgtgtgcttggtcagcatatctgagacgatgttccgctgtcccagaccagtccagtaatggacgggccagtgtgcgtagtcgtcttccggcttgtccggcgcatgtttggtgaaccggtggggtaaggttggtgtgcccaacgcccgtactttggtgatacctcaagaccacccaggaatgccagggaggtaccccgcttcacagcgggatctgaccctgggctaattgtctacggtggttcttcttgcttccacttctttctactgttcgccacc 352 CaprinegtggccacgcccgggccaccgatacttcccttcactccttcgggactgttggggaggaacacaacKobuvirusagggctcccctgttttcccattccttcccccttttcccaaccccaaccgccgtatctggtggcggcaa5Δ40 gacacacgggtctttccctctaaagcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcgggagtgctcccacccaactgttgtaagcctgtccaacgcgtcgtcctggcaagactatgacgtcgcatgttccgctgcggatgccgaccgggtaaccggttccccagtgtgtgtagtgcgatcttccaggtcctcctggttggcgttgtccagaaactgcttcaggtaagtggggtgtgcccaatccctacaaaggttgattctttcaccaccttaggaatgctccggaggtaccccagcaacagctgggatctgaccggaggctaattgtctacgggtggtgtttcctttttcttttcacacaactctactgctgacaactcactgactatccacttgctctcttgtgcctttctgctctggttcaagttccttgattgtttttgactgcttttcactgcttttcttctcacaatccttgctcagttcaaagtc 353 CaprinegtggccacgcccgggccaccgatacttcccttcactccttcgggactgttggggaggaacacaacKobuvirusagggctcccctgttttcccattccttcccccttttcccaaccccaaccgccgtatctggtggcggcaa5Δ40/3Δ122gacacacgggtctttccctctaaagcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcgggagtgctcccacccaactgttgtaagcctgtccaacgcgtcgtcctggcaagactatgacgtcgcatgttccgctgcggatgccgaccgggtaaccggttccccagtgtgtgtagtgcgatcttccaggtcctcctggttggcgttgtccagaaactgcttcaggtaagtggggtgtgcccaatccctacaaaggttgattctttcaccaccttaggaatgctccggaggtaccccagcaacagctgggatctgaccggaggctaattgtctacgggtggtgtttcctttttcttttcacacaactaaagtc 354 CaprinegtggccacgcccgggccaccgatacttcccttcactccttcgggactgttggggaggaacacaacKobuvirusagggctcccctgttttcccattccttcccccttttcccaaccccaaccgccgtatctggtggcggcaa5Δ40/3Δ86_gacacacgggtctttccctctaaagcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcgDistalggagtgctcccacccaactgttgtaagcctgtccaacgcgtcgtcctggcaagactatgacgtcgcatgaccgctgcggatgccgaccgggtaaccggttccccagtgtgtgtagtgcgatcttccaggtcctcctggttggcgttgtccagaaactgcttcaggtaagtggggtgtgcccaatccctacaaaggttgattctttcaccaccttaggaatgctccggaggtaccccagcaacagctgggatctgaccggaggctaattgtctacgggtggtgtttcctttttcttttcacacaactctactgctgacaactcactgactatccacttgctctaaagtc 355 CaprinegtggccacgcccgggccaccgatacttcccttcactccttcgggactgttggggaggaacacaacKobuvirusagggctcccctgttttcccattccttcccctttttcccaaccccaaccgccgtatctggtggcggcaa5Δ40/3Δgacacacgggtctttccctctaaagcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcg122_Kozakggagtgctcccacccaactgttgtaagcctgtccaacgcgtcgtcctggcaagactatgacgtcgcatgttccgctgcggatgccgaccgggtaaccggttccccagtgtgtgtagtgcgatcttccaggtcctcctggttggcgttgtccagaaactgcttcaggtaagtggggtgtgcccaatccctacaaaggttgattctttcaccaccttaggaatgctccggaggtaccccagcaacagctgggatctgaccggaggctaattgtctacgggtggtgtttcctttttcttttcacacaactgccacc 356 CaprinegtggccacgcccgggccaccgatacttcccttcactccttcgggactgttggggaggaacacaacKobuvirusagggctcccctgttttcccattccttcccccttttcccaaccccaaccgccgtatctggtggcggcaa5Δ440/3Δ86gacacacgggtctttccctctaaagcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcgProximalggagtgctcccacccaactgttgtaagcctgtccaacgcgtcgtcctggcaagactatgacgtcgcatgttccgctgcggatgccgaccgggtaaccggttccccagtgtgtgtagtgcgatcttccaggtcctcctggttggcgttgtccagaaactgcttcttggtaagtggggtgtgcccaatccctacaaaggttgattctttcaccaccttaggaatgctccggaggtaccccagcaacagctgggatctgaccggaggctaattgtctacgggtggtgtttcctttttcttttcacacaactttcactgcttttcttctcacaatccttgctcagttcaaagtc 357 Parabovirustgaaccgttacgcaccactcagttggtgtttggtggcaccaatgatggaacaaaaggctacaccacttgggctacggcccgcgccaccttgtggcgcaaagacattagaagaatagcataccgcccactagggccctgcagccagcagggtaacgggcaagcacttctgtctccccggtagaacggtataggctgtacccacggccgaaaactgaactatcgttacccgactccgtacttcgcaaagcttagtaggaaactggaaagttcgagttattgacccggagtgttccccccactccagaaacgcgtgatgagggttgccaccccgaccatggcgacatggtgggcatccctgcgctggcacgcggcctctaagaggataactcgctcctactggtaaccgaagagccccgtgagctacggtttattcctccgcctccctgaatgcggctaatcctaacccatgagcagttgccatagatccatatggtggactgtcgtaacgcgtaagttgtgggcggaaccgactactttgggatggcgtgtttccttgttttctccatttgttgttgtatggtgacaagttatagatctcgatctatagcgtttcttgagagatttccaaacatttattcaagtcgtacaattcttgtgtttaagcagtacagtgtaagg 358 Parabovirusaacaaaaggctacaccacttgggctacggcccgcgccaccttgtggcgcaaagacattagaaga 5Δ48atagcataccgcccactagggccctgcagccagcagggtaacgggcaagcacttctgtctccccggtagaacggtataggctgtacccacggccgaaaactgaactatcgttacccgactccgtacttcgcaaagcttagtaggaaactggaaagttcgagttattgacccggagtgttccccccactccagaaacgcgtgatgagggttgccaccccgaccatggcgacatggtgggcatccctgcgctggcacgcggcctctaagaggataactcgctcctactggtaaccgaagagccccgtgagctacggtttattcctccgcctccctgaatgcggctaatcctaacccatgagcagttgccatagatccatatggtggactgtcgtaacgcgtaagttgtgggcggaaccgactactttgggatggcgtgtttccttgttttctccatttgttgttgtatggtgacaagttatagatctcgatctatagcgtttcttgagagatttccaaacatttattcaagtcgtacaattcttgtgtttaagcagtacagtgtaagg 359 Parabovirustgggctacggcccgcgccaccttgtggcgcaaagacattagaagaatagcataccgcccactag 5Δ67ggccctgcagccagcagggtaacgggcaagcacttctgtctccccggtagaacggtataggctgtacccacggccgaaaactgaactatcgttacccgactccgtacttcgcaaagcttagtaggaaactggaaagttcgagttattgacccggagtgttccccccactccagaaacgcgtgatgagggttgccaccccgaccatggcgacatggtgggcatccctgcgctggcacgcggcctctaagaggataactcgctcctactggtaaccgaagagccccgtgagctacggtttattcctccgcctccctgaatgcggctaatcctaacccatgagcagttgccatagatccatatggtggactgtcgtaacgcgtaagttgtgggcggaaccgactactttgggatggcgtgtttccttgttttctccatttgttgttgtatggtgacaagttatagatctcgatctatagcgtttcttgagagatttccaaacatttattcaagtcgtacaattcttgtgtttaagcagtacagtgtaagg 360 Parabovirustgaaccgttacgcaccactcagttggtgtttggtggcaccaatgatggaacaaaaggctacaccac 3Δ60ttgggctacggcccgcgccaccttgtggcgcaaagacattagaagaatagcataccgcccactagggccctgcagccagcagggtaacgggcaagcacttctgtctccccggtagaacggtataggctgtacccacggccgaaaactgaactatcgttacccgactccgtacttcgcaaagcttagtaggaaactggaaagttcgagttattgacccggagtgttccccccactccagaaacgcgtgatgagggttgccaccccgaccatggcgacatggtgggcatccctgcgctggcacgcggcctctaagaggataactcgctcctactggtaaccgaagagccccgtgagctacggtttattcctccgcctccctgaatgcggctaatcctaacccatgagcagttgccatagatccatatggtggactgtcgtaacgcgtaagttgtgggcggaaccgactactttgggatggcgtgtttccttgttttctccatttgttgttgtatggtgacaagttatagatctcgatctatagcgtttgtaagg 361 ApodemustttgaaaggggtgcggatatcatggcgtttctcgccatgatatccgcacattgcaaacccatattgcaPicornavirustacccactgggtatgcattatggggaggcccctttcacccctccccccccaattaccttttccccctctagtaaccatacgctttactcagcgtaactactccgggttacgtgatgaagaagaggctacggagattctcgggctacggccctggagccactccggctcctaaagatttagaagtttgagcacacccgcccactagggccccccatccaggggggcaacgggcaagcacttctgtttccccggtatgatctgataggctgtaaccacggctgaaacagagattatcgttatccgcttcactacttcgagaagcctagtaatgatgggtgaaattgaatccgttgatccggtgtctcccccacaccagaaactcatgatgagggttgccatcccggctacggcgacgtagcgggcatccctgcgctggcatgaggcctcttaggaggacggatgatatggatcttgtcgtgaagagcctattgagctagtgtcgactcctccgcccccgtgaatgcggctaatcctaaccccggagcaggtgggtccaatccagggcctggcctgtcgtaatgcgtaagtctgggacggaaccgactactttcgggaaggcgtgtttccatttgttcattatttgtgtgtttatggtgacaactctgggtaaacgttctattgcgtttattgagagattcccaacaattgaacaaacgagaactacctgttttattaaatttacacagagaagaattaca 362 ApodemuscccctccccccccaattaccttttccccctctagtaaccatacgctttactcagcgtaactactccgggPicornavirusttacgtgatgaagaagaggctacggagattctcgggctacggccctggagccactccggctccta 5Δ105aagatttagaagtttgagcacacccgcccactagggccccccatccaggggggcaacgggcaagcacttctgtttccccggtatgatctgataggctgtaaccacggctgaaacagagattatcgttatccgcttcactacttcgagaagcctagtaatgatgggtgaaattgaatccgttgatccggtgtctcccccacaccagaaactcatgatgagggttgccatcccggctacggcgacgtagcgggcatccctgcgctggcatgaggcctcttaggaggacggatgatatggatcttgtcgtgaagagcctattgagctagtgtcgactcctccgcccccgtgaatgcggctaatcctaaccccggagcaggtgggtccaatccagggcctggcctgtcgtaatgcgtaagtctgggacggaaccgactactttcgggaaggcgtgtttccatttgttcattatttgtgtgtttatggtgacaactctgggtaaacgttctattgcgtttattgagagattcccaacaattgaacaaacgagaactacctgttttattaaatttacacagagaagaattaca 363 ApodemusattctcgggctacggccctggagccactccggctcctaaagatttagaagtttgagcacacccgccPicornaviruscactagggccccccatccaggggggcaacgggcaagcacttctgtttccccggtatgatctgata 5Δ201ggctgtaaccacggctgaaacagagattatcgttatccgcttcactacttcgagaagcctagtaatgatgggtgaaattgaatccgttgatccggtgtctcccccacaccagaaactcatgatgagggttgccatcccggctacggcgacgtagcgggcatccctgcgctggcatgaggcctcttaggaggacggatgatatggatcttgtcgtgaagagcctattgagctagtgtcgactcctccgcccccgtgaatgcggctaatcctaaccccggagcaggtgggtccaatccagggcctggcctgtcgtaatgcgtaagtctgggacggaaccgactactttcgggaaggcgtgtttccatttgttcattatttgtgtgtttatggtgacaactctgggtaaacgttctattgcgtttattgagagattcccaacaattgaacaaacgagaactacctgttttattaaatttacacagagaagaattaca 364 KobuvirusttcacaccctctttccggtggtccggacccagaccaccgttactccattcagctacttcggtacctgSZAL6ttcggaggaattaaacgggcaccctacccaagggttacatgggaccatattcctcctcccctgtaactttaagtttgtgcccgtattcttgactccaggcggatgttgtgtcgcccgtcctgtgaacaaacagctagacactttcctcccctccctctgggctgctccggcagtccactccctccccccagcgtaacatgccccgctggagtgatgcacctggaagtcgtggacgtgggttagtaacttcggtgaaaacccactataatgacaactggttgacccccacactcaaaggactcgagtctttctcccttaaggctagcccggccacatgaatttgcagctggcaactagtgagtccaccatgtcccgcaacctcggctgcggagtgctgttccccaagcgtatgccttccttctgtaagagtgcgcctggcaagcacatctgagaagtcgttccgctgcgtcgtgccaacctggcgacaggtgacccagtgtgcgtagacttcttccggattcgtccggctcttctctaggaaacatgcgtgtaaggttcatgtgccaaagccctgcgcgcggtgttcttctactgccctaggaatgtgccgcaggtacccctacttcggtagggatctgagcggtagctaattgtctacgggtagtttcatttccatcttctcttcaggtcgacatc 365 KobuvirusttgactccaggcggatgttgtgtcgcccgtcctgtgaacaaacagctagacactttcctcccctccctSZAL6 5Δ158ctgggctgctccggcagtccactccctccccccagcgtaacatgccccgctggagtgatgcacctggaagtcgtggacgtgggttagtaacttcggtgaaaacccactataatgacaactggttgacccccacactcaaaggactcgagtctttctcccttaaggctagcccggccacatgaatttgcagctggcaactagtgagtccaccatgtcccgcaacctcggctgcggagtgctgttccccaagcgtatgccttccttctgtaagagtgcgcctggcaagcacatctgagaagtcgttccgctgcgtcgtgccaacctggcgacaggtgacccagtgtgcgtagacttcttccggattcgtccggctcttctctaggaaacatgcgtgtaaggttcatgtgccaaagccctgcgcgcggtgttcttctactgccctaggaatgtgccgcaggtacccctacttcggtagggatctgagcggtagctaattgtctacgggtagtttcatttccatcttctcttcaggtcgacatc 366 KobuvirusgaattaaacgggcaccctacccaagggttacatgggaccatattcctcctcccctgtaactttaagttSZAL6 5Δ76ttgtgcccgtattcttgactccaggcggatgttgtgtcgcccgtcctgtgaacaaacagctagacactttcctcccctccctctgggctgctccggcagtccactccctccccccagcgtaacatgccccgctggagtgatgcacctggaagtcgtggacgtgggttagtaacttcggtgaaaacccactataatgacaactggttgacccccacactcaaaggactcgagtctttctcccttaaggctagcccggccacatgaatttgcagctggcaactagtgagtccaccatgtcccgcaacctcggctgcggagtgctgttccccaagcgtatgccttccttctgtaagagtgcgcctggcaagcacatctgagaagtcgttccgctgcgtcgtgccaacctggcgacaggtgacccagtgtgtgtagacttcttccggattcgtccggctcttctctaggaaacatgcgtgtaaggttcatgtgccaaagccctgcgcgcggtgttcttctactgccctaggaatgtgccgcaggtacccctacttcggtagggatctgagcggtagctaattgtctacgggtagtttcatttccatcttctcttcaggtcgacatc 367 KobuvirustttcacaccctcttttccggtggtccggacccagaccaccgttactccattcagctacttcggtacctgSZAL6 3Δ37ttcggaggaattaaacgggcaccctacccaagggttacatgggaccatattcctcctcccctgtaactttaagttttgtgcccgtattcttgactccaggcggatgttgtgtcgcccgtcctgtgaacaaacagctagacactttcctcccctccctctgggctgctccggcagtccactccctccccccagcgtaacatgccccgctggagtgatgcacctggaagtcgtggacgtgggttagtaacttcggtgaaaacccactataatgacaactggttgacccccacactcaaaggactcgagtctttctcccttaaggctagcccggccacatgaatttgcagctggcaactagtgagtccaccatgtcccgcaacctcggctgcggagtgctgttccccaagcgtatgccttccttctgtaagagtgcgcctggcaagcacatctgagaagtcgttccgctgcgtcgtgccaacctggcgacaggtgacccagtgtgcgtagacttcttccggattcgtccggctcttctctaggaaacatgcgtgtaaggttcatgtgccaaagccctgcgcgcggtgttcttctactgccctaggaatgtgccgcaggtacccctacttcggtagggatctgagcggtagctaattggacatc 368 SalivirustctgtcctcaccccatcttcccttctttcctgcaccgttacgcttactcgcatgtgcattgagtggtgcaSZ1 cgtgcttgaacaaacagctacactcacatgggggcgggttttcccgccctgcggcctctcgcgaggcccacccctccccttcctcccataactacagtgctttggtaggtaagcatcctgatcccccgcggaagctgctcacgtggcaactgtggggacccagacaggttatcaaaggcacccggtctttccgccttcaggagtatccctgctagtgaattctagtagggctctgcttggtgccaacctcccccaaatgcgcgctgcgggagtgctcttccccaactcaccctagtatcctctcatgtgtgtgcttggtcagcatatctgagacgatgttccgctgtcccagaccagtccagtaatggacgggccagtgtgcgtagtcgtcttccggcttgtccggcgcatgtttggtgaaccggtggggtaaggttggtgtgcccaacgcccgtactttggtgatacctcaagaccacccaggaatgccagggaggtaccccgcttcacagcgggatctgaccctgggctaattgtctacggtggttcttcttgcttccacttctttctactgttcatg 369 Crohivirusgtataagagacaggtgtttgccttgtcttcggactggcatcttgggaccaaccccccttttccccagc Bcatgggttaaatggcaataaaggacgtaacaactttgtaaccattaagctttgtaattttgtaaccactaagctttgtgcacataatgtaaccatcaagcttgttagtcccagcaggaggtttgcatgcttgtagccgaaatggggctcgaccccccatagtaggatacttgattttgcattccattgtggacctgcaaactctacacatagaggctttgtcttgcatctaaacacctgagtacagtgtgtacctagaccctatagtacgggaggaccgtttgtttcctcaataaccctacataataggctaggtgggcatgcccaatttgcaagatcccagactgggggtcggtctgggcagggttagatccctgttagctactgcctgatagggtggtgctcaaccatgtgtagtttaaattgagctgttcatatacc 370 CrohivirusccccccttttccccagccatgggttaaatggcaataaaggacgtaacaactttgtaaccattaagcttB 5Δ51tgtaattttgtaaccactaagctttgtgcacataatgtaaccatcaagcttgttagtcccagcaggaggtttgcatgcttgtagccgaaatggggctcgaccccccatagtaggatacttgattttgcattccattgtggacctgcaaactctacacatagaggctttgtcttgcatctaaacacctgagtacagtgtgtacctagaccctatagtacgggaggaccgtttgtttcctcaataaccctacataataggctaggtgggcatgcccaatttgcaagatcccagactgggggtcggtctgggcagggttagatccctgttagctactgcctgatagggtggtgctcaaccatgtgtagtttaaattgagctgttcatatacc 371 CVB3ttaaaacagcctgtgggttgatcccacccacagggcccattgggcgctagcactctggtatcacggtacctttgtgcgcctgttttataccccctcccccaactgtaacttagaagtaacacacaccgatcaacagtcagcgtggcacaccagccacgttttgatcaagcacttctgttaccccggactgagtatcaatagactgctcacgcggttgaaggagaaagcgttcgttatccggccaactacttcgaaaaacctagtaacaccgtggaagttgcagagtgtttcgctcagcactaccccagtgtagatcaggtcgatgagtcaccgcattccccacgggcgaccgtggcggtggctgcgttggcggcctgcccatggggaaacccatgggacgctctaatacagacatggtgcgaagagtctattgagctagaggtagtcctccggcccctgaatgcggctaatcctaactgcggagcacacaccctcaagccagagggcagtgtgtcgtaacgggcaactctgcagcggaaccgactactttgggtgtccgtgtttcattttaacctatactggctgcttatggtgacaattgagagattgttaccatatagctattggattggccatccggtgaccaatagagctattatatatctctttgttgggtttataccacttagcttgaaagaggttaaaacattacaattcattgttaagttgaatacagcaaa 372 CVB3 3Δ91ttaaaacagcctgtgggttgatcccacccacagggcccattgggcgctagcactctggtatcacggtacctttgtgcgcctgttttataccccctcccccaactgtaacttagaagtaacacacaccgatcaacagtcagcgtggcacaccagccacgttttgatcaagcacttctgttaccccggactgagtatcaatagactgctcacgcggttgaaggagaaagcgttcgttatccggccaactacttcgaaaaacctagtaacaccgtggaagttgcagagtgtttcgctcagcactaccccagtgtagatcaggtcgatgagtcaccgcattccccacgggcgaccgtggcggtggctgcgttggcggcctgcccatggggaaacccatgggacgctctaatacagacatggtgcgaagagtctattgagctagttggtagtcctccggcccctgaatgcggctaatcctaactgcggagcacacaccctcaagccagagggcagtgtgtcgtaacgggcaactctgcagcggaaccgactactttgggtgtccgtgtttcattttattcctatactggctgcttatggtgacaattgagagattgttaccatatagctattggattggccatccggtgaagcaaa 373 SAFVcacttatttaattcggccttttgtgacaagcccctcggtgaaagaacctctctcttttcgacgtggttggaattgccatcatttccgacgaaagtgctatcatgcctccccgattatgtgatgttttctgccctgctgggcggagcattctcgggttgagaaaccttgaatctttttctttggaaccttggttcccccggtctaagccgcttggaatatgacagggttattttcttgatcttatttctacttttgcgggttctatccgtaaaaagggtacgtgctgccccttccttctctggagaattcacacggcggtctttccgtctctcaacaagtgtgaatgcagcatgccggaaacggtgaagaaaacagttttctgtggaaatttagagtgcacatcgaaacagctgtagcgacctcacagtagcagcggactcccctcttggcgacaagagcctctgcggccaaaagccccgtggataagatccactgctgtgagcggtgcaaccccagcaccctggttcgatgatcattctctatggaaccagaaaatggttttctcaagccctccggtagagaagccaagaatgtcctgaaggtaccccgcgtgcgggatctgatcaggagaccaattggcggtgctttacactgtcactttggtttaaaaattgtcacagcttctccaaaccaagtggtcttggttttccaattttgttga 374 SAFV 5Δ46cctctctcttttcgacgtggttggaattgccatcatttccgacgaaagtgctatcatgcctccccgattatgtgatgttttctgccctgctgggcggagcattctcgggttgagaaaccttgaatctttttctttggaaccttggttcccccggtctaagccgcttggaatatgacagggttattttcttgatcttatttctacttttgcgggttctatccgtaaaaagggtacgtgctgccccttccttctctggagaattcacacggcggtctttccgtctctcaacaagtgtgaatgcagcatgccggaaacggtgaagaaaacagttttctgtggaaatttagagtgcacatcgaaacagctgtagcgacctcacagtagcagcggactcccctcttggcgacaagagcctctgcggccaaaagccccgtggataagatccactgctgtgagcggtgcaaccccagcaccctggttcgatgatcattctctatggaaccagaaaatggttttctcaagccctccggtagagaagccaagaatgtcctgaaggtaccccgcgtgcgggatctgatcaggagaccaattggcggtgctttacactgtcactttggtttaaaaattgtcacagcttctccaaaccaagtggtcttggttttccaattttgttga 375SAFV 5Δ93gtgctatcatgcctccccgattatgtgatgttttctgccctgctgggcggagcattctcgggttgagaaaccttgaatctttttctttggaaccttggttcccccggtctaagccgcttggaatatgacagggttattttcttgatcttatttctacttttgcgggttctatccgtaaaaagggtacgtgctgccccttccttctctggagaattcacacggcggtctttccgtctctcaacaagtgtgaatgcagcatgccggaaacggtgaagaaaacagttttctgtggaaatttagagtgcacatcgaaacagctgtagcgacctcacagtagcagcggactcccctcttggcgacaagagcctctgcggccaaaagccccgtggataagatccactgctgtgagcggtgcaaccccagcaccctggttcgatgatcattctctatggaaccagaaaatggttttctcaagccctccggtagagaagccaagaatgtcctgaaggtaccccgcgtgcgggatctgatcaggagaccaattggcggtgctttacactgtcactttggtttaaaaattgtcacagcttctccaaaccaagtggtcttggttttccaattttgttga 376 SAFV 3Δ47cacttatttaattcggccttttgtgacaagcccctcggtgaaagaacctctctcttttcgacgtggttggaattgccatcatttccgacgaaagtgctatcatgcctccccgattatgtgatgttttctgccctgctgggcggagcattctcgggttgagaaaccttgaatctttttctttggaaccttggttcccccggtctaagccgcttggaatatgacagggttattttcttgatcttatttctacttttgcgggttctatccgtaaaaagggtacgtgctgccctttccttctctggagaattcacacggcggtctttccgtctctcaacaagtgtgaatgcagcatgccggaaacggtgaagaaaacagttttctgtggaaatttagagtgcacatcgaaacagctgtagcgacctcacagtagcagcggactcccctcttggcgacaagagcctctgcggccaaaagccccgtggataagatccactgctgtgagcggtgcaaccccagcaccctggttcgatgatcattctctatggaaccagaaaatggttttctcaagccctccggtagagaagccaagaatgtcctgaaggtaccccgcgtgcgggatctgatcaggagaccaattggcggtgctttacactgtcactttggtttaatgttga 377SAFV Kozakcacttatttaattcggccttttgtgacaagcccctcggtgaaagaacctctctcttttcgacgtggttggaattgccatcatttccgacgaaagtgctatcatgcctccccgattatgtgatgttttctgccctgctgggcggagcattctcgggttgagaaaccttgaatattttctttggaaccttggttcccccggtctaagccgcttggaatatgacagggttattttcttgatcttatttctacttttgcgggttctatccgtaaaaagggtacgtgctgccccttccttctctggagaattcacacggcggtctttccgtctctcaacaagtgtgaatgcagcatgccggaaacggtgaagaaaacagttttctgtggaaatttagagtgcacatcgaaacagctgtagcgacctcacagtagcagcggactcccctcttggcgacaagagcctctgcggccaaaagccccgtggataagatccactgctgtgagcggtgcaaccccagcaccctggttcgatgatcattctctatggaaccagaaaatggttttctcaagccctccggtagagaagccaagaatgtcctgaaggtaccccgcgtgcgggatctgatcaggagaccaattggcggtgctttacactgtcactttggtttaaaaattgtcacagcttctccaaaccaagtggtcttggttttccaattttgttgaccgcc 378 GLuc CKgtggccacgcccgggccaccgatacttcccttcactccttcgggactgttggggaggaacacaac dCTG1agggctcccctgttttcccattccttcccccttttcccaaccccaaccgccgtatctggtggcggcaagacacacgggtctttccctctaaagcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcgggagtgctcccacccaactgttgtaagcctgtccaacgcgtcgtcctggcaagactatgacgtcgcatgttccgctgcggatgccgaccgggtaaccggttccccagtgtgtgtagtgcgatcttccaggtcctcctggttggcgttgtccagaaactgcttcaggtaagtggggtgtgcccaatccctacaaaggttgattctttcaccaccttaggaatgctccggaggtaccccagcaacagctgggatctgaccggaggctaattgtctacgggtggtgtttcctttttcttttcacacaactctacGTctgacaactcactgactatccacttgctctaaagtc 379 GLuc CKgtggccacgcccgggccaccgatacttcccttcactccttcgggactgttggggaggaacacaacdCTG1_2agggctcccctgttttcccattccttcccccttttcccaaccccaaccgccgtatctggtggcggcaagacacacgggtctttccctctaaagcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcgggagtgctcccacccaactgttgtaagcctgtccaacgcgtcgtcctggcaagactatgacgtcgcatgttccgctgcggatgccgaccgggtaaccggttccccagtgtgtgtagtgcgatcttccaggtcctcctggttggcgttgtccagaaactgcttcaggtaagtggggtgtgcccaatccctacaaaggttgattctttcaccaccttaggaatgctccggaggtaccccagcaacagctgggatctgaccggaggctaattgtctacgggtggtgtttcctttttcttttcacacaactctacGTcGTacaactcactgactatccacttgctctaaagtc 380 GLuc CKgtggccacgcccgggccaccgatacttcccttcactccttcgggactgttggggaggaacacaacdCTG1_2_3agggctcccctgttttcccctttccttcccccttttcccaaccccaaccgccgtatctggtggcggcaagacacacgggtctttccctctaaagcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcgggagtgctcccacccaactgttgtaagcctgtccaacgcgtcgtcctggcaagactatgacgtcgcatgttccgctgcggatgccgaccgggtaaccggttccccagtgtgtgtagtgcgatcttccaggtcctcctggttggcgttgtccagaaactgcttcaggtaagtggggtgtgcccaatccctacaaaggttgattctttcaccaccttaggaatgctccggaggtaccccagcaacagctgggatctgaccggaggctaattgtctacgggtggtgtttcctttttcttttcacacaactctacGTcGTacaactcacGTactatccacttgctctaaagtc 381 GLuc CK dAllgtggccacgcccgggccaccgatacttcccttcactccttcgggactgttggggaggaacacaacagggctcccctgttttcccattccttcccccttttcccaaccccaaccgccgtatctggtggcggcaagacacacgggtctttccctctaaagcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcgggagtgctcccacccaactgttgtaagcctgtccaacgcgtcgtcctggcaagactatgacgtcgcatgttccgctgcggatgccgaccgggtaaccggttccccagtgtgtgtagtgcgatcttccaggtcctcctggttggcgttgtccagaaactgcttcaggtaagtggggtgtgcccaatccctacaaaggttgattctttcaccaccttaggaatgctccggaggtaccccagcaacagctgggatctgaccggaggctaattgtctacgggtggtgtttcctttttcttttcacacaactctcGTcGTacaactcacGTactaCTcactGTctctaaagtc 382 CK SZ1-L1Sgggggtggggggggcctcggccccctcaccctcttttccggtggccacgcccgggccaccgatacttcccttcactccttcgggactgttggggaggaacacaacagggctcccctgttttcccattccttcccccttttcccaaccccaaccgccgtatctggtggcggcaagacacacgggtctttccctctaaagcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcgggagtgctcccacccaactgttgtaagcctgtccaacgcgtcgtcctggcaagactatgacgtcgcatgttccgctgcggatgccgaccgggtaaccggttccccagtgtgtgtagtgcgtcttccaggtcctcctggttggcgttgtccagaactgcttcaggtaagtggggtgtgcccaatccctacaaaggttgaaccacccaggaatgccagggaggtaccccgcttcacagcgggatctgaccctgggctaattgtctacggtggttcttcttgcttccacttctttctactgttcgccacc 383 CK Aichi Scangggggtggggggggcctcggccccctcaccctcttttccggtggccacgcccgggccaccgat (AV-S)acttcccttcactccttcgggactgttggggaggaacacaacagggctcccctgttttcccattccttcccccttttcccaaccccaaccgccgtatctggtggcggcaagacacacgggtctttccctctaaagcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcgggagtgctcccacccaactgttgtaagcctgtccaacgcgtcgtcctggcaagactatgacgtcgcatgttccgctgcggatgccgaccgggtaaccggttccccagtgtgtgtagtgcgtcttccaggtcctcctggttggcgttgtccagaactgcttcaggtaagtggggtgtgcccaatccctacaaaggttgattctttcaccaccttaggaatgctccggaggtaccccagcaacagctgggatctgaccggaggctaattgtctacgggtggtgtttcatttccaatccttttatgtcggagtc 384 CK Aichi Loopgggggtggggggggcctcggccccctcaccctcttttccggtggccacgcccgggccaccgat (AV-L1)acttcccttcactccttcgggactgttggggaggaacacaacagggctcccctgttttcccattccttcccccttttcccaaccccaaccgccgtatctggtggcggcaagacacacgggtctttccctctaaagcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcgggagtgctcccacccaactgttgtaagcctgtccaacgcgtcgtcctggcaagactatgacgtcgcatgttccgctgcggatgccgaccgggtaaccggttccccagtgtgtgtagtgcgtcttccaggtcctcctggttggcgttgtccagaactgcttcaggtaagtggggtgtgcccaatccctacaaaggttgaactgccctaggaatgccaggcaggtaccccacctccgggtgggatctgagcctgggctaattgtctacgggtagttttcctttttcttttcacacaactctactgctgacaactcactgactatccacttgctctcttgtgcctttctgctctggttcaagttccttgattgtttttgactgcttttcactgcttttcttctcacaatccttgctcagttcaaagtc 385CK SZ1-L2gggggtggggggggcctcggccccctcaccctcttttccggtggccacgcccgggccaccgatacttcccttcactccttcgggactgttggggaggaacacaacagggctcccctgttttcccattccttcccccttttcccaaccccaaccgccgtatctggtggcggcaagacacacgggtctttccctctaaaaagcctgtccaacgcctgatcccccgcggaagctgctcacgtggcaactgtggggacccagacaggttatcaaaggcacccggtctttccgccttcaggagtatccctgctagtgaattctagtagggctctgcttgcgttgtccagaaactgcttcaggtaagtggggtgtgcccaatccctacaaaggttgattctttcaccaccttaggaatgctccggaggtaccccagcaacagctgggatctgaccggaggctaattgtctacgggtggtgtttcctttttcttttcacacaactctactgctgacaactcactgactatccacttgctctcttgtgcctttctgctctggttcaagttccttgattgtttttgactgcttttcactgctttcttctcacaatccttgctcagttcaaagtc 386 CK Aichigggggtggggggggcctcggccccctcaccctcttttccggtggccacgcccgggccaccgat TriLoopacttcccttcactccttcgggactgttggggaggaacacaacagggctcccctgttttcccattcctt(AV-L2)cccccttttcccaaccccaaccgccgtatctggtggcggcaagacacacgggtctttccctctaaagcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcgggagtgctcccacccaactgttgtaagcctgtccaacgcatgtgcctggcaagcatatctgagaaggtgttccgctgtggctgccaacctggtgacaggtgccccagtgtgcgtaaccttcttccgtctccggacggtgcgttgtccagaaactgcttcaggtaagtggggtgtgcccaatccctacaaaggttgattctttcaccaccttaggaatgctccggaggtaccccagcaacagctgggatctgaccggaggctaattgtctacgggtggtgtttcctttttcttttcacacaactctactgctgacaactcactgactatccacttgctctcttgtgcctttctgctctggttcaagttccttgattgtttttgactgcttttcactgcttttcttctcacaatccttgctcagttcaaagtc 387CK Scan gggggtggggggggcctcggccccctcaccctcttttccggtggccacgcccgggccaccgatDeletion acttcccttcactccttcgggactgttggggaggaacacaacagggctcccctgttttcccattcctt(ΔS) cccccttttcccaaccccaaccgccgtatctggtggcggcaagacacacgggtctttccctctaaagcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcgggagtgctcccacccaactgttgtaagcctgtccaacgcgtcgtcctggcaagactatgacgtcgcatgttccgctgcggatgccgaccgggtaaccggttccccagtgtgtgtagtgcgatcttccaggtcctcctggttggcgttgtccagaaactgcttcaggtaagtggggtgtgcccaatccctacaaaggttgattctttcaccaccttaggaatgctccggaggtaccccagcaacagctgggatctgaccggaggctaattgtctacgggtggtg 388 CK LoopgggggtggggggggcctcggccccctcaccctcttttccggtggccacgcccgggccaccgatDeletion acttcccttcactccttcgggactgttggggaggaacacaacagggctcccctgttttcccattcctt(ΔL1) cccccttttcccaaccccaaccgccgtatctggtggcggcaagacacacgggtctttccctctaaagcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcgggagtgctcccacccaactgttgtaagcctgtccaacgcgtcgtcctggcaagactatgacgtcgcatgttccgctgcggatgccgaccgggtaaccggttccccagtgtgtgtagtgcgatcttccaggtcctcctggttggcgttgtccagaaactgcttcaggtaagtggggtgtgcccaatccctacaaaggttgatttcctttttcttttcacacaactctactgctgacaactcactgactatccacttgctctcttgtgcctttctgctctggttcaagttccttgattgtttttgactgcttttcactgcttttcttctcacaatccttgctcagttcaaagtc 389 CK TriloopgggggtggggggggcctcggccccctcaccctcttttccggtggccacgcccgggccaccgatDeletion acttcccttcactccttcgggactgttggggaggaacacaacagggctcccctgttttcccattcctt(ΔL2) cccccttttcccaaccccaaccgccgtatctggtggcggcaagacacacgggtctttccctctaaagcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcgggagtgctcccacccaactgttgtaagcctgtccaacgcgcgttgtccagaaactgcttcaggtaagtggggtgtgcccaatccctacaaaggttgattctttcaccaccttaggaatgctccggaggtaccccagcaacagctgggatctgaccggaggctaattgtctacgggtggtgtttcctttttcttttcacacaactctactgctgacaactcactgactatccacttgctctcttgtgcctttctgctctggttcaagttccttgattgtttttgactgcttttcactgcttttcttctcacaatccttgctcagttcaaagtc 413 RhPVgataaaagaacctataatcccttcgcacaccgcgtcacaccgcgctatatgctgctcattaggaattacggctccttttttgtggatacaatctcttgtatacgatatacttattgttaatttcattgacctttacgcaatcctgcgtaaatgctggtatagggtgtacttcggatttccgagcctatattggttttgaaaggacctttaagtccctactatactacattgtactagcgtaggccacgtaggcccgtaagatattataactattttattatattttattcaccccccacattaatcccagaaaagctttataactataagtaagccgtgccgaaacgttaatcggtcgctagttgcgtaacaactgttagtttaattttccaaaaattatttttcacaatttttagttaagattttagcttgccttaagcagtctttatatcttctgtatattattttaaagtttataggagcaaagttcgctttactcgcaatagctattttatttattttaggaatattatcacctcgtaattatttaattataacattagctttatctatttata 414 Halastaviarvacttgattctaaccttgccgtatggtgccctaacgggttcatttaatcatgcgatgagggttgctatacc(1x mut)gcatccattctaaggcgattcaatgcttcatttaggaattagttgacgattaaaaggtacccccacaaaaacaaaaccaatcttacttgattttcgttttaactgaccactgcgatcccaaattttcgccttcttatcaaagtatgttgtgttctttgggtgtacaacctgagaacttgtctacaactacatattactcgaggaagaaattcggtttaagccgtgccttctcacgtttagtatatctatctGgacacaccttcttcatcttctaatccccatctagtctcctgatcagagacgtcgttattaacaaataaccccccttgttaataagagacaaagtacaatcaagctaagttctcttggagttcctgtaggaacttagccattgtgatagagtcataagtctatgtgcatagacagctctagctcaccatttccttcccaacccatcttttcatcagcttaactctatgaatccgatgcaaaaaccattctaacatcttatggtgctttccaagccaaatgagagctcactcttttgagccgctatttaatggacaataaacgttttatagtgtacatcatattgtaaaaacaaa 415 Osciviruscctcggtccctctttccgtcgccgcccacgacgttaaatgcggtgttgtggtgcttaggtgccacaccactgctatttgggtccccctttcccctatatatgtttgtttgtttatttcaatttcttgaggattggcacctccttatgccaaatctaaatcgtggaggatcccaggctttctggtctttaacagaactccacgtccaggtcatagaaactggttggtaggctgcctgagtagtccatttgctagtagtcccttgtgaacagggtggctcccgtttactgctggtattcccggtgtaggtcgccatggtggtaacaccatcctgcattgtgtgtgaaccagtaccgcaaggatagcaaggtatgaacacttgtggacgaaatggtaagtgatcaattcactttcatggccggaaggtcacgtggcaatcatgccacccaggtaccctcctctgggaggatctgagggtgggctaagcagaccctgccatgtggctgaacttttcccttattgttttactttgtaacatttatagttgtgttagtgatttgtgtgttgtgcccttgtgagctatatccagtataagttcgcagctagaagttaatccttcgacatcggctgtattggaa 416 Cadiciviruscaccaacccttgacctgtaatgtcagtggacagagtgctcctctgttcccggttaccgtgttccagg Bacacgattgtaatcctgcgcctcaccagcgctgcgtgcacgtctgcataaggaaacgtgccttccccatgtctctatcaattctttggtgagtgaccgccctagttgctcatcctatgggattcttctctcatgggttctttgtggcatgcgaatgtcaccttaattggaggtctttaattagatatcctttcttcatctttgatatgagtgtcggaatttgattcctagtctctgcaaaacaaccccacttgatgaattcaacttttcaaccgcacaaacataatcaggtttttaaattgaatgtttctaaattctaaatttagtttatttaagtagtttgccatcttgactcgatgtaaaattgtcatacaagtcttcttttcttttctttacactttgaagtttgcacttagcagtcgttctgcacagctttcgagttttgtttgatcgacatcgcaacttccacccacctctctttttctagtgttgaatgcggctaatcctaacccgagagcaataaacccaggtttattgtcgtaacgcgcaagtcttggacggaaccgactatacacacacctctttaccctttagtacacccttggtacg 417 PSIV (2xgcaaaatgggtacgtagttaaccactgcgtatcaggattgcaggccacgaagggtatttgcatatctmut forttctatgcggtattacggcttaaaacccgttgtatcttgtTgtttgactgcctgtatcactagtggccattXba1)ttatttaggttagagacccctgatagtaggagagttacaaactctttaaaaattgttgaccccggaaaagatggtgacccctgtaagtagttgatcAagaagatctatgcgctggcatagtaatccagtgtttcctgttttaggatgacctctgaaagtagatgaccgtggaaagtcacgtagtgccccaataagcacgtttgggcagcgtgcgctatcacaaggcttgatctccgaggagccccttgttttagctggctggaagccaatgatcttaagtagataagtgctgttgcttgtagttcaacagaaagctttgagtacgtctttcttgcgagaaagaacacatgcattcttatgctctcaattctattatttttattttgggcgaaaggaaagctctcacgcgagtacgaatagccaaccctttat 418 PSIV IGRGCTGACTATGTGATCTTATTAAAATTAGGTTAAATTTCGAGGTTAAAAATAGTTTTAATATTGCTATAGTCTTAGAGGTCTTGTATATTTATACTTACCACACAAGATGGACCGGAGCAGCCC TCCAATATCTAGTGTACCCTCG 419PV Mahoney ATGAGTCTGGACATCCCTCACCGGTGACGGTGGTCCAGGCTGCGTTGGCGGCCTACCTATGGCTAACGCCATGGGACGCTAGTTGTGAACAAGGTGTGAAGAGCCTATTGAGCTACATAAGAATCCTCCGGCCCCTGAATGCGGCTAATCCCAACCTCGGAGCAGGTGGTCACAAACCAGTGATTGGCCTGTCGTAACGCGCAAGTCCGTGGCGGAACCGACTACTTTGGGTGTCCGTGTTTCCTTTTATTTTATTGTGGCTGCTTATGGTGACAATCACAGATTG TTATCATAAAGCGAATTGGATTGGCC420 REV A GGGGTCGCCGTCCTACACATTGTTGTGACGTGCGGCCCAGATTCGAATCTGTAATAAAAGCTTTTTCTTCTATATCCTCAGATTGGCAGTGAGAGGAGATTTTGTTCGTGGTGTTGGCTGGCCTACTGGGTGGGGTAGGGATCCGGACTGAATCCGTAGTATTTCGGTACAACATTTGGGGGCTCGTCCGGGATTCCTCCCCATCGGCAGAGGTGCCTACTGTTTCTTCGAACTCCGGCGCCGGTAAGTAAGTACTTGATTTTGGTACCTCGCGAGGGTTTGGGAGGATCGGAGTGGCGGGACGCTGCCGGGAAGCTCCACCTCCGCTCAGCAGGGGACGCCCTGGTCTGAGCTCTGTGGTATCTGATTGTTGTTGAACCGTCTCTAAGACGGTGATACTATAAGTCGTGGTTTGTGTGTTTGTTTGTTACCTTGTGTTTGTTCGTCACTTGTCGACAGCGCCCTGCGAATTGGTGTACCCACACCGCGCGGCTTGCGAATAATACTTTGGAGAGTCTTTTGCCTCCAGTGTCTTCCGTTTGTACTCGTCCTCCTCTCCCTCTCCGGCCGGGATGGG 421 Tropivirustgtcgcatgttgccaacatcaaaattctgggagagtcgcgaactccttaacactgccttgcctcgac Aggagccgttgttatagtgtcgacgggatacaaacattaaactaaacccacttgcctcgacggaaccccttaccttttatttttttatatagtatgaaagtgaatcttgtatgaatgttcatagaaaactgcaaatgagtaccacgtctaacatgagagaatgatactggagaaatccaagtttagaagtcactacgaatcccagcggaaacaagggaattctgagcttctaataggcgtttaagactatttgcaaaattctggtgcgtaagtgatattttcattgcgtagaacgctggtaaccactccggctagtataagcattgttagtcacttattatgaaactccacactatcctttctggagaagcacacaaacttacatggtaaagctagaccattatcttaagcggtgagtacactgcaaccttgtaacaatgcttgtatgactactttttgtatatcttgagcaatattgttgaggtggacatgtccaaaggtaatgttgttgggaatggaggggtccattttcccgtgcacgtagtgtactagtattgggtgatagccttgcggcggatcaaccatgtattttaatccgttgactttcac 422Symapivirusttgggaaatccccaatgcttctttcaacaccgcctgactatgcggtggcgcttcggctcaaacaact Aagtcacttccccctcttaactactacccaagacttctaactacccttacctacttatttgtctaaatttcaaacttttattctcacgcgtcttataaacatcttttctatttgttatggtatgttttgtgatttgtgtggtgtatttcatttaatgggatctagtggaccgtgccccggttgggtatccgctccctttaaatgtttgcaagcactcttgacattataacctatcatttagtttacttgtttgtatgatcgtatttctgaatcgtaacatttatgcaattctttctcgccgagacttgtctaggagataaagttcctgcatatttagtgttacggttgtataatggagacttagatagcttcacactgaggacgctttttcgctatccttttgacctgattcaggccagtgtggagttaatgattgtatggatgggccctacaatttgtctaagacttggtgatagcctcgcggccgctcgccatttatacaactgaatagcggttgaaactctct 423 Sakobuvirustcacgcgcttttccggtggtcacccaccgttagggagcgccagcgttcgcgcttccgctaccaggt AgacacactcctttcccctcccccattcccgttcccatcctctggactggtttctcctcacgattgaccaFFUP1 gcagctgggagctgttaccagacgttggacagtaagtcccggatgcactatagggctggtggcta(1x mut)gtgcttggtaagcactcaacgccatacctaatgtgtacctcggcttgccctcctggtcgtggtgaccggctgtttctcttcccttggctcCagacgggctggtgtcctaccaccaccgttgcatgcagacctccccctgcgcactcgaacgccctgtcccagcagggttagtatgtgctgtgcagatctgcatgtgacaccccatccactggtagagcaggaagttgccctagctaacgcggcaagtattactttccgctacacgtccttgagattcctcggacctctggaactagggtgactgtgggcttgggaaaacccaccttggtcctgtactgcctgatagggtcgcggctggccgaccagtggatgtagccagttgttttgggat 424 Rosaviruscagggagatctccatgaataatcttttccaccctctttagcgtctatgctattgaggacgggttggag CccccgttgacccagcgtcagagtgtgtcggtagcaggctttctgctctcgccccatgccggccacaNFSM6Fcctcccattagtgatgtgaaggttgtaagttacatgtgaaaaggtttctaataattgagctgaatgtagcgattacctaaggtgagcggattcccccacgtggtaacacgtgcctctcaggccaaaagccaaggtgttaaaagcaccccttaggtaggccactaccccgtggcctcagttctcttagaagattcacttagtagtgtgtgcactggcaactcttaagcagagctagtgagtgggctaaggatgccctgaaggtacccgcaggtaacgttaagacactgtggatctgatcaggggctcgagtgctgaagctttacagaggtagctcgagttaaaaaacgtctatgcccctcccccacgggagtgggggacccccacaccaattttagatt gcact425 Rosavirusccaggcatggcgttaaacatgcattcccttcccctagtaacctcccttcgccccttccccacgttgta 2ccccctccgagatggctgctaaggcgcttgctgctacagcagtctcgtgtttcgggtgttataagtgGA7403ctttcttttccactccactccctgcctatggggagcggaacggccttgtctcggtcgttgcttcttgcagatcttcacccctccaggctttctggactcgccaggggtggagtagtaggcgcactgtctaagtgaaggtagcagtgttgttggcgaagagttgtggacctactttgagtttgtagcgatcatccagagctagcggatctccccacgcggtaacgcgtgcctctaggcccaaaaggcacggtgttcacagcaccctttggatggcgggggtgcccccctccgcacttaaagtagaaaaacagcttagtagtcaaataacatggctttcctcaagcattcagtgctcatgggactgaaggatgcccagaaggtacccgcaggcaacgataagctcactgtggatctgatctggggccctgggccaggtgctatacacctggttaaaaccaaatctggtagtcagggttaaaaaacgtctaagtcccacccccccggggacggggggttcccttaaaccctcaactgacacc 426 Rhimaviruscgaattccggacatctcctttcgggggcgagcgtcaccgtgcccctcatggaggcaactgtgcctc Ataatcggtgacccactgagaaaattttctttctacgtggctaaacaatgcaactttataataacacaaatttaatgcttaatcttaacaccaaagatttgaacatatgtttggaaagtggcacacttcaaacattgcatagttgctaggggtgaagtccctttaaggggttgcagaggatctttcctctttatgagcggctaggagtatcttcttgatattatgtggtcgtgcaactcacttcccagatgtatgacggtgtactaagcgattggaactagtcataacctctttgaattttggtattgcgagtctagcagggggatatttaccgctaaagggtgacacactcgtgagggtggcctttggtgtgtgtatatttattccgcccatcttgcatggggtgctaaaattctaatgctgtgaaataaccattttctgaatacattctctacatttggagtcaaatatgaggaatgccactcaggtacccttgacatgatcttggatctgagagtgggctaattatctaattatttggcgactttctaaaatcttctgtttttagtggtgacaatttatggttataaa 427 RafivirusgtgtccgggaagcgactcaagcttttgactgagtctctacaccttcatccgtaacatctttaagtttatgLPXYC222841tgcctatggacctctagtgcactgccatcaccgggggtgtattggactggtttttccacaatccattcatcctgaggaattttggctttgttactaggatggtcccaccacacgcttatctgtgcctattgtgtcaaccatgttcttaagtagttgtgcccgtgggtgagtagataaccacaacaatccgataaagcatctcgcaaggatgtgagtaatggagtgtatgtgctacagagacccacaacctgaaccaagagagacacagtgaggattgtaaagggggaactctttgaaagggcatgtcccgcaattcctactgactgacaccgggggttggtgtcggtggattttagcaaatcctgttactgggtgatagccttgtgcacttcacttggttcttgtataagtgctgta 428 RafivirustgcgaatttattcgcacagtctcttttcccccatcttgtgtgtgtgatggggtaagccgcagagtaataWHWGGF74766cctactctgctgcaaacacactcactcttttctatctactttatatcatgtaataataagtagggaacatattcaattcatattgttcatctcactgaacccgcatgaaggactgcattgcatatcctggacgaagtgacgtggaatatttggacatttatggattggacaccattacgctttgtgcctctacggagatgtaaccataatcttaagtagtagtaccccagcacaagaggataaagtggcatacacgacaacgggtgttgctcgcaccttagtaatgtggatgttcacccttggagcgtgctgaaactctgtgggtaaagacacacattagtacaaatgtgggggaactcactgaaagggcatgtcccgtgtactggtgtgccggaaagtgggggtcgctttctggagaacttagtagttcttgttattgggtgatagccttgcggcggatcaactcacagttttaatccgttgttttgcat 429 PoecivirusactacacaatcgcaacacgcgcaagtttgtagtttgattggcgtgcaaatgtcaaatcaagcatataBCCH-449acacaatttggtggctgttggtgtttgttataggaattttggttgtgttgaaattgtggatgtgtaggaaatatgcacaattacgtcagcgtcaggagttttataacctggcgcaacaccaaaatggtcttcgcgctttaacatcaccagcgaggtgtaaacaaattgaagttgaattagatcgtgtataggccagggaaccatccctcccaacgccacatcttgtggggaagttgggataatggtgggtctatatgaattggtctgtagacccacagtgaagagtgaatagtatgcttgcggttccatttgttaatggtctagcatgggtgggggcggcaaccccgtgaggggttccccactggccaaaagcccaggggttagtcatttcaaccaaggaagctggtaacctggtgacctgaacttgagtggtgagacccccttgctagagtgtgtaaaccgattgtaagcattttgtttgcttagtatctgtggtataagcagtcaattttgtataggctcaaggctgtggtagttagtagatgcccggaaggttattactgatccggggaccgtgactatacattaggtaaaccggtttaaaaacc 430Megirivirusttcgggacactggatgggcgacttggtggggctgccactctatcttgacctttcgttactgactttcg ALYgatctctgactcctccttgtctcttgcgtttggtccacggacggactaattggaatgtttactggctaagcctcgttctgaaataccctagccaatgggttgtagtaggatcctggtgtttccattaaacctcttccgaccatagtagctagagttatggctgtgtaggatgtgggtaagaccgctttttgcgtatctcccacaagacaccggattatggatgtgtccgctggataaggctcgaaacctcccaactgaaggtggtgctgaaatattgcaagcctaggttgtgtagaggcaagtagatgcctgccgcgacattcgtcttccgcccttttgggttagtagtgtacctacatggacgtggggctgggaatccccaccttgcataacactggttgatagacctgcggctggtcaagttactatggtataaccagttgaaatggct 431 Megirivirusgcttggcaacctcatatcgttactctgccgaccagtctgggtcgtgtggccacacaatgggattcgtt Ectgttgtgtagagtcacatggcattactgggctgatcggtggggatccgttgccacccctaaacccttacatttactggactgcttttcttggccccggaatgattcgctcacccgcgatgaggactgttgttcttattatggcaggattacgcgtctggtccgcgtaaggactaattcctatgtttatacgttactaccttgttctgaacggtgggcgccaccccgcctagtaggatcctggcttatcgtgtagacctctagggaccacattagctagagtgtaggctgctatggatggagtagtgacccctttttgggtatcactctctaagactccggaatgtgtcatagtacgctggaaatccttacttgtttttccatgagggggaggtggtgctgaaatattgcaagccacccctcggttaaaacagtttggtgccgcttatgccatattaccgccccttgtagttgggctgtttttgcagctccgggttagtagagtaccatagtggacgcggtgttgggaatcaccgccttggctgcacactgcttgatagagctgcggctggtcaagctaattgtggtataaccagttgatttggcat 432Megirivirusttcccgaccggtctggcaaaccggacggttatcctggttagatgtctgatggttgctggaacgtggt Cggctactgctgccaccttctggcttcctttaatgggcatctagctgggttctttgccacaatccatcttactctcttacccattttctattacccagacttgttgaaactggtaaagttgacctactggcttcgttttgagactattctggtgttggtggacactctttccacaagtagattgtatggagttcatgctcgttttgaaccgggaatggcacaacccgtagtaggatcttgcctctgccatactaatctgcgcctgttgcttttagactatgggctgctaaggatgacattggaacccctttttggatattccatgtcaagtcaactgtttcatctggtgtacgctggaaatccttgttccgaggtcttgtctggaggtggtgctgaaatattgcaagccacaggcagttccttggacttggtgccgctatcagatgctacaccctctatgggcaaatgttgaaccttagtggacgcgtgagatgggaatccacgccggccatagactggctgataagctcgcggctgatcgagttgcaacagtaatcagttgatttgccact 433 Ludopivirustagacccccacctagcccttttccccgtcagtggggggcttactcactgggcatctgttaatctggcctaactagattgacaccactcccttggaacgtaactccacgctaactcactggctctacgcacagacacacggtctttctgctatccccggggaagataccagatggcgaccggctgtcccagcggcctagtagctactcgggttgagtacccaccacggttttgacgcctgctaaaattcaagagacagaggtaggggtgcttagtgtgtgggggaagttcccacaagcgaggcaaagcattgctccctcgcgtcaccgggtgcaaggtaaattggctggacttccgctctacccttgctactcgccctcttcggagggttcgaagtgacactaggtatacgcatggttgggaaaccatgcctggcctactactgggtgatagcctggcggcgggtccgtctcttggcttatacccgttgatttgggat 434 Livupivirustatctacatggggatccaggctgtatggaatgtctgtcttaacaagcactataccagaaagatccacccaaagtggtgggactgggactgtgaggtgagaaatcccgaaaccagccttctcaagcgtcggacgatctttctgttttagtgaacaccttgccttttaaatggatgacaacaccccttcagcaaatcgcaatctgaaatcccaaaagactgtttagccgaactctggtaatcactccggagaagtaggatacgcagcccctgtggactcttgatttcaggactcaaggtagctagagctggaacttcatggaatgacaaaggaatatatgcacattgtgcgctttcctggccttgtagcccgtcgtgaggatatgtcgttgggaatcgacatcttagtccagtactgcttgatagagtgtcggctggcacagttacctgagaataagtcagttgtacttaacatgaacaaaaaaaataactaccacaactaccacaatctaccaatacttgaattatgctgaatctcgtacagtaaaaacgttccgtggaaggacaagtattgaagtgcggttacatcatccgatacgcgctggatc cctca435 Aichiviruscacccatacacccccacccccttttctgtaactcaagtatgtgtgctcgtaatcttgactcccacgga AatggatcgatccgctggagaacaaactgctagatccacatcctccctccccttgggaggacctcggFSS693tcctcccacatcctccctccagcctgacgtatcacaggctgtgtgaagcccccgcgaaagctgctcacgtggcaattgtgggtccccccttcatcaagacaccaggtctttcctccttaaggctagccccgatgtgtgaattcacattgggcaactagtggtgtcactgtgcgctcccaatctcggccgcggagtgctgttccccaagccaaacccctggcccttcactatgtgcctggcaagcatatctgagaaggtgttccgctgtggctgccagcctggtaacaggtgccccagtgtgcgtaaccttcttccgtctccggacggtagtgattggttaagatttggtgtaaggttcatgtgccaacgccctgtgcgggatgaaacctctactgccctaggaatgccaggcaggtaccccaccttcgggtgggatctgagcctgggctaattgtctacgggtagtttcatttccaattcttttatgctggagtc 436 Aichivirustactccattcagcttcttcggaacctgttcggaggaattaaacgggcacccatactcccccccaccc KVGHcccttttgtaactaagtatgtgtgctcgtgaccttgactcccacggaacggaccgatccgttggtgaacaaacagctaggtccacatcctcctttcccctgggagggtccccgccctcccacatcccccccccagcctgacgtgtcacaggctgtgtgaagcccccgcgaaagctgctcacgtggcaattgtgggtccccccttcatcaagacaccaggtctttcctccttaaggctagccccggcgtgtgaactcacgttgggcaactagtggtgtcactgtgcgctcccaatctcggccgcggagtgctgttccccaagccaaacccctggcccttcactatgtgcctggcaagcacacctgagaaggtgttccgctgtggctgccagcctggtaacaggtgccccagtgtgcgtaaccttcttccgtcttcggacggtggtgattggttaagatttggtgtaaggttcatgtgccaacgccctgtgcgggatgaaacctctactgccctaggaatgccaggcaggtaccccaccttcgggtgggatctgagcctgggctaattgtctacgggtggtttcatttccaattctttcatgtcggagtc 437 Aichivirustactccattcagcttcttcggaacctgttcggaggaattaaacgggcacccatacacccccaccccc DVttttctgcaacttaagtatgtgtgctcgtaatcttgactcccacggaacggatcgatccgctggagaacaaactgctagatccacatcctcccttcccctgggaggaccccggtcctcccacatcctccccccagcctgacgtaacacaggctgtgtgaagtccccgcgaaagctgctcacgtggcaattgtgggtccccccttcaccaagacaccaggtctttcctccttaaggctagccccgatgtgtgaattcacattgggcaactagtggtgtcactgtgcgctcccaatctcggccgcggagtgctgttccccaagccaaacccctggcccttcactatgtgcctggcaagcatatctgagaaggtgttccgctgtggctgccagcctggtaacaggtgccccagtgtgcgtaaccttcttccgtctccggacggtagtgattggttaagatttggtgtaaggttcatgtgccaacgccctgtgcgggatgaaacctctactgccctaggaatgccaggcaggtaccccaccttcgggtgggatctgagcctgggctaattgtctacgggtagtttcatttccaattcttttatgtcggagtc 438 MurinegtaacttcaagtgtgtgtgctcgtaatcttgactcctgccggaatgccgcccggttcagtgaacaaaKobuvirus 1cagctaggcaagtccctcccttcccctgtggtcggttctcaccggccaccatccctcccccagcctgacgtgttacaggctgtgcaaagcccccgcgaaagctgctcacgtggcaattgtgggtcccccctttgtcaagacaccgagtctttctcccttaaggctagcccggtcccacgaacgtggaactggcaactagtggtgtcactacacgcctccgacctcggacgcggagtgctgttccccaagctgtaaccctgacccaagactgtgctgcctggcaagcaccgtctgggaagatgttccgctgtggctgccaaacctggtaacaggtgccccagtgtgtgtagtcttcctccagtctccggactggcagtcttgtgtaaagatgcagtgtaaggttcaagtgccaaatccctggaaggagtgaccctctactgccctaggaatgctgtgcaggtacccccaacttcggttggggatctgagcacaggctaattgtctacgggtagtttcatttcccatcctctcttttttggcatc 439 PorcinetttgaaaagggggtgggggggcctcggccccctcaccctcttttccggtggccacccgcccgggKobuvirusccaccgttactccactccactccttcgggactggtttggaggaacataacagggcttcccatccctg K-30tttacccttactccactcacccctccccttgaccaaccctatccacaccccactgactgactcctttggatcttgacctcggaatgcctacttgacctcccacttgcctctcccttttcggattgccggtggtgcctggcggaaaaagcacaagtgtgttgttggctaccaaactcctacccgacaaaggtgcgtgtccgcgtgctgagtaatgggataggagatgccaataacaggctcgcccatgagtagagcatggactgcggtgcatgtgacttcggtcaccaggggcatagcattgctcacccctgaatcaagtcatcgagatttctctgacctctgaagtgcactgtggttgcgtggctgggaatccacgcttgaccatgtactgcttgatagagtcgcggctggccgactcatgggttaaagtcagttgacaagacac 440 PorcineccaccgttacttcactccactccctcgggactggtttggaggagcataacagggcttcccatccctgKobuvirusttcaccctcaataccacccaccctttccctcaaccatccctatccacaccccactgactgattcccttg XXgattttgacctcagaacgcctacttgacctcccacttgcctttcccttctcggattgccggtggtgcctggcggaaaaagcacaagtgtgttgcaggctaccaaactcctacccgacaaaggtacgtgtccgcgtgctgagtaatgggataggagatgcctacaacaggctcgcccatgagtagagcatggactgcggtgcatgtgacttcggtcaccacgggcatagcattgctcacccgtgaatcaagtcattgagattcctctgacctctgaagtgcactgtggttgcgtggctgggaatccacgcttgaccatgtactgcttgatagagtcgcggctggccgactcatgggttaaagtcagttgataagacac 441 CaprinegggggtggggggggcctcggccccctcaccctcttttccggtggccacgcccgggccaccgatKobuvirusacttcccttcactccttcgggactgttggggaggaacacaacagggctcccctgttttcccattcctt12Q108cccccttttcccaaccccaaccgccgtatctggtggcggcaagacacacgggtctttccctctaaagcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcgggagtgctcccacccaactgttgtaagcctgtccaacgcgtcgtcctggcaagactatgacgtcgcatgttccgctgcggatgccgaccgggtaaccggttccccagtgtgtgtagtgcgatcttccaggtcctcctggttggcgttgtccagaaactgcttcaggtaagtggggtgtgcccaatccctacaaaggttgattctttcaccaccttaggaatgctccggaggtaccccagcaacagctgggatctgaccggaggctaattgtctacgggtggtgtttcctttttcttttcacacaactctactgctgacaactcactgactatccacttgctctcttgtgcctttctgctctggttcaagttccttgattgtttttgactgcttttcactgcttttcttctcacaatccttgctcagttcaaagtc442 RabbitgggctataaatatgggcattcctcttcccccttccccttttgaagatgagtgcgcatattcttgactccgKobuviruscctggattggccgcccaaggcgtgaacaagcagctaggccaccatgacactgcggtggtgtccgaacccgcgggtgccttcacgggcacctgtggtatgtaggactcccaccgtggtcttccctttccccctcaatctttccccctggttcgactaacgggaccagtgctggaacctgtccggtgaacggtatagcaggcccccccggcagaaacacccggtgcttaccccttaaggctagcccccacctttgaatttggttggggcaactagtgggtgtacagttggcgtgaaccctccggtctaggagtgctcttgcccaatcctctgtgtgtgccttgcagtagggactggcaatccttcgcgtaggtgatccgctgtgccatgccatcctggcgacaggaggcccagtgtgcgcaacctacgtcccttctgggtgctgcattgcattacctttggagtaagcttggtgtgccgaaaccccagggtttacgtaccactcgtggtgtgaggaatgtgccgcaggtaccccatccttgaggtgggatctgagcggtagctaattgtctagcaccactttcttccttttttctttgctggtcacg 443 Aalivirusttgaaagggggtgctcagggtagctccctgagctcttccctccttccctcatcaacgtctggcccacgatacgggccacctttcaatcttaactaactatccctttaatctatttggattttctggtttagaataatttggaacacataattggattatcttttaggattgtggataggatttgttcgggatatcactcccttcctgtgctaacacatattctaattccctcctttgtctattatctcttggaggtggtgctgaaatattgcaagccacttgagtgtatagatgaagtaggctcaagatgaatgttgtgttactcaaggcaagtgtagctatcactaagatattggtaacgtgaaacggattaccggtagtagcgtgatcttccgtcttagtgctctagtgactagaggacaacgacatggcatcacatatcttaaccctccagttttggcatccgggacagaatgggctggatatccgctttctttctggggtatgtgatgggtggtattggggtaaccaccttgaccatgacgctcgataagagtgaccgcctgatcattgaaacctctagtataaaattcaggctgaaatc 444 Grusopivirustgcctgagtaggattgtgaatttaggtatgagagggttagccaacccattctgaaccataatagatac Agtcaatctgaatccatctaaatctatctcttaggcagtggtgctgaaatattgcaagctactagggatagacgtgatctgattcaagaacctatctaatgtggtgatgagaaggctaggtttatccatagtaatcccttgttctgaacaggcaatgcacatgctctagtaggatctcgggctctgcgattggctctaaaccgaccaatccaggtagaggcactaagtgtaggacttgccaaaatgtattacatgctggtaccgactcactagtctggaaactccacactgaaagtgactggggggggccccatcacatttgtgctactgcttgatagagttgcggctggtcaacttggattggtataaccagttgaa 445 Grusopivirusgccatccgtaggttctggtaaggttccatcaactgttggggcgctagttgctatgaccgcattcacg Bgacggatgatttatagtatcacccaatccgggcacaacttctttagccacttcttccacattactaagggctctcttgccgagtttcaacgtctagtccacgacacggaccttcctacttttctatcttcttattttctctactaaattggtatctggtactgaagatatgcggattgtgattttgtgcctgtctaaactaaccctattctagggttaggtgggtaccatatactaatggtgaacaggattacctatgtatccattagtccctatggatctggcgacccacaaactcatgttcatagagaggctaagctgagtgctcgccgaataagcattgcttcaggtgccgactattgtctggaaaccactcagtgatagctataggggggggccccgtagcatctgccttactgcctgatagggtggcggctggtccatgaacatgcagtaaccagttgacttgac 446 Yanchengccttagggtctggaatgcgtcctttctgggcacttccacaatcctaaggtaattttcaacgccagcgaosbeckstggagcgatatccaaagaacctttatgttttagttcgtcttgtatgtttataaaatataaaattgggattagrenadiergcaacccacaaaaacatttttgttattctcaccacatgagagggtggttaaacctcttcgtaacctatcanchovyttgcttgattggctacttgggctagatttaggacaacctctttagcaagcctaaattactcctcgtacapicornavirusgcacctggtaacaggcgtacctggaggttacggtggcgctaacttggacttctcgttaattcgtgcaagtttaaatgatgcctattttgaatacaagaaagtatgatagtaacttagggcgtgaagttccgcttaacataaggcagtataagtactaagataaggtgtaagacctaccttaataactgttgtcttttctcatggtctttccgtgggagcccttgctaggggtaaagttaagtattctcaaataatttttcattcaaactctttctctctgtttt 447 TurkeyccactcgcacttcctggatagtgcgttagatatgcccgacatatcgcttccaggaccaaagcccccGalliviruscttttctctttcccaaccagcttcgccactcaagctgtaattccatgtccggtctttccggccttagtatcM176atggaaatgtggtcgtgctcaaatgaaattgagttgacattgatcaatgaaagttgcactgaactttgctaaactggctagcgccacctggtgtgtgccgttggtctcctcacatggtaacatgtgccaacgggcccgaaaggctagtgggcaattaccgctccaagggaggggtacccaccccgacctgaacagcggtaatgaagctcacctcccaggctctgaccccgagaagtttagttatttagtaggtgtaattagtacttgtgattggtcaatagatagtagatgaaacgttatggatgaatgagtagaccccctgaaggtaccccattacatgggatctgatcagggccacattctgcgtgtctccccgcacttgtggttaaaaccatgaaagttcatcccaaacaatcttttcctcttctttttcttttagtggtgacaacctactggattggtgattaccaatctgtactagtgttgtattaagacttgttgtgtggagaaaatggactctttcaagaagatttttg 448Falcovirustaaaaggggacgcggtgtggcagctttggctgtcatgccgtgttctccttttaccccaaggactagc A1cttgggggttttccaaattcctttccctgtaggctttacttctctttatctatcttttctgtaactaagttttgcctattctaaaaatattttagaatgtgtttggatgtaactaagtttgtgcctgccctaaaaatattttagggcttgtttggataacctcgtcccttgtgttcagtgccgcacaatttgctaggcactgttcacttcctttgtttgtccattatgtatgctaaggtatgaattccatcatatgcttagcctctacatgcataatcttattccctccctggtgcaaactacgcccccaacatatgtgaatcttttaagcatattcctgaccccacacatatatatgtgttctcgtgaattcccccaccgtgaggtggtcacttggacgtggtgtgtgtcacacagcatatatatgatgcaggatgttgtttttaagataagcatatgtccttagtgctttgcatcatttcctccacaccccgtgaatgcggctaatcttaaccctgttgggtccgtgggtaaaccaacccattaaccacaggacggaaccgactactttcgggagtgtgtgtttctttttcttcttttgtcact 449 Tremovirusttcaaatggcccctgggttgatacccagtggtcatttggacactttggtaaggaggtgtaattatcctt Bcccatgtggaacctagtgcttaggtttactttatatgttctttgtttgtcctttgtactttctatcgggcaatcttgttgttcaatacaatatgtatttgaactgcctaagataaattcagttttcaaccaacccctctcttggggttgtgtctttctttctttcttatatcctcttaagctgacttacttgctaatccgactcctcgtcaacgggagggtaaagcagtatcactagggtattgtgatgtaggagaaaaagtaagtagagatagtgcatgtaacgaaagtgacttggtactttaaactctcttaatcccaaagtgtggtattggtcatgttggagtaggctacgggtgaaactccttcacatttagtaatgtgttcacacgctaacgctacggtagatgacagactaggtcttattctcaacgtagggggacgggtgtatgttcatgattagccacatattaaggttttgaggggctgagtcatataagtatgtgcattaatttctggtactggtccctggggactggcccttttctaggttgattttagtttccccaatttttaaaaactaatgagatttacgac 450 DidelphistctttggtctggggaactaaaataccagacccgcgtttgcctagcgatataggctttaattgttgtttgtauritacattgtgcgtttgatatgtgttttaatgtaaataataattctagcaggttctagacttgatcatgtcctctttHAVaaggcactcatttcaacttgctatctttcttttcttccttggttctccctacaccaaatgcactggccgctgcgcccggcggggtcaaccacatgattagcatgtggctgtaggtgttgaaggctgggacatgaacatcaatggaatagtgcgcatgcttactggggtccattgaagtagtgggatctttctattggggtaggctacgggtgaaaccccttaggttaatactcatattgagagataccttggataggttaactgtgctggatatggttgagtttaacgacaaaaagccatcaacagctgtggacagaacctcatccttagattgctcactatggatatgtgctctgggcgtgtttcttgcatgatggccattggtcaattcatgcctgggccaatgtaggattagccttaaattactttttaaaagtagcctcatttagctggactaatggtggggcgtatgatcctgcatttggcctctggggtaatcaggggcatttaggtttccacataatagcaaat 451 HepatovirusgcaaggggtggttttaaccttgcacgcgtttaccgtgcgttaacggttttccatgtttgtatgtcttgtttG1gtattatgtgttttgtaaatattaattcctgcaggttcagggttctttaatcatgttgggctgtacccacactcaacttttggccataagtgagtttcttaacgaaccttttaacacaggatgttattagggcccaatattttccctgaggccttctttggcctctattttttccccttttctatctccttgtattccgggctcacgtgatgccaatggactgacccatgcgcccgtgggggttaactactggagtagccagtagctgtaggtgctaaaagtcacgtacgtgtaagactggacgagacctctcagctataactgaaagtagtaagtatgtctgaacttcttgaaggggtaggctacgggtgaaaccccttaggttaatactcatattgagagatacctctgataggtgaaggtttccggtagaggtgagtttaacgacaaagcctctcaacggatgtgggcccacctcatcagcaagatgctttcatacccaataccgtaggggctgggttgttgagacagtcccaagcgtccctcccgcaaggttgtaggggtactcaggggcatttaggtttccacaattaaacaaataca 452 Hepatovirusctttggatgcccatagtgcgggggtataaataccgcactccctttagctgttccgagggtatcggaa Dcctatatgtttgttttctgtctgtctgtcagctttatgtgtgctcgtcccctttagggcactcatttcagcttgctttcattcttttcttccccggttctcaccttaccggaggcactggccgttgcgcccggcggggtcaacctagtgattagcactaggctgtaggtgtctaaagtggtgacattaagacttggtaactgatttcagcactgttaactgatgttggggatgacttgattgatcttctggaaggggtaggctacgggtgaaaccccttatcttaataccactatgtagagatagattcagtaggttaagggcagtggataaggttgagttcattttggacaataaaccttcaacactggtggacccaatctcactgaccagatgctttcttgactgatccttcagaggggtgattcttctgaataggttgccttgacactgatgcctgagacccattgggtcgggccttaaatcatggaactccactggactttcatggcctagcttctgccttagacagactctggggccccacgaccctctgggcccttcggggtactcaggggcatttaggtttttccacaattaaaagagtta 453HepatovirusgtcatgtttctctttaagaacactcaattttggccataagtgagactcttgtcgaacctttcatgtcaggH2accatgttagggccattatccttttccctggggcattcttcttgcccctgtttcatctttctatcatctttcttccgggctctcacaatgccaatggagcgaccgatgcgcacgtcggggttaacccatggattagccatgggctgtagctgctaaaagttgtgactcctgaagcatactatcaatggtagtagatgtaactgaaacactgaagcttctctgatcttgaaagaagggtaggctacgggtgaaacccttcaggttaatactcatattgagagatacctttggtaggttaacgttggcggataatgttgagtttaacgacaataaacattcaacgcctgtgggcgaacctcaccaatttcatgctttgaagtgaatgtgcgtagggtctctatcggagatgctatgtggatggtgccctccctggaaacaggttgtaggggtactcaggtgcacttaggtttccacattttaaagatttttc 454 Hepatovirusggctgcctgtgtctcaggggtaagtactggggccgcgttgaccgtgcggtacggttatgcttttaga Ittaggatgtccgtctgtccggcactctcttttgcttaaaatggccttaaatccatgggaggcgtaaccatgggccctttgttacctagacatgattgcattgggggccgtccttggggcttaggccccagccatttctcttgactcgtctaagagtttacttcatccttttctttactttattttccaggctctcagcatgccgacggctctgaccactgcgcccggtggggttaactgcatgattagcatgcagctgtaggagttaaaagtgctgacaggccaattctgacgtaagtccactctatattaacttgatcaagtaaggttgattgatctttgtgagagggtaggctacgggtgaaaccctctaggttaatactcatattgagagatacctccagaaggtgaaggttggcggatattggtgagttcttttaggacaaaaacctttcaacgcctgtgggcccacctcactggcacaatgctttcatccccaattgtgatgggtagtttggactgaaatcaggagtaacctgccctacgagtttaggggtagttcaggggtatttaggcttccacatttgatagagtttatgagagtgagcc 455Hepatovirusttcaaaagccccagcggggtttcattaccccgctgtggcttttggacttccctaggatggggaagta Caattaccatcctcgcgtttgccgtgcgttaacggctacttttcttctagctgtagaagtaaaattcagcatgttttatgtttgtttgtcttgtttgttatatacttttatacactcctacaaatgcacatgaagaacagtttgtagagattaacaaacgcttagctgaacctaggtggtgaatctagtagtaagataagtagaggaagctataccttaagttggttgggccctcgtgtttgctctataaacaaaaccaagtgagtagagtggatgaacagtactaaatccctgagtacagggaacctcacaggtgtgatacacttatgtctatgtgacctggttggaggttgggcgtgccctatgatactggagtgggagatcttttggggaacccacgttttcacactgcctgatagggtcttgccgagagactcacttgtttcggctgtacttgtaac 456 Fipivirustgcgggtaaactcccgcatgtgtgaatgaggcgatgtcccaggaactaactgccgatcctggtttta Aactacgatccgtatttgttactaatgcgatatccccccattgtttgcctccatgttgttttcaacgcttttggccttgagtgttatcaagtgttttagcgacatagtgggaagctacggctgcgtccccatttttgagtggcgacccagttttagtggccactctgtccctgaactgcgctataatgtgaatttatgttcacaaaaacggactgatgtaactgttaatgactaaggaatagtacctcactgaagtatcaagaccccgttcgagcggtgtacatatatggatggaaaccagtctgagtcatctcgaatactaatcaatgagggatgtcgagtaagcatatcatgaaccacatagaatagtggggtttcggggttagaggctctctgcagcaatgtatctctaacaccatggccgaaatgagagatagagaccacgatgtttgtgtgtaagtaatgatgtgtggaaagaaaattctgaatgttggtatgatatcagtctaaggggagtggctcacctaagagctacccaaacatttcacagcagacaacataacgtactgagagtagttggaaggttccagaaatcagt 457 Fipiviruscgcggttaaacccgcgccaaccttctttcagccgcgtctgagtagcgcggttagtcctgatacacagt Cttcctgttgggtactgtgtcttcgggtgaatgctcttgtgtgaatgttttaggctgtttaagggaagcgtttccccgtgcgctgtgagggtttctcacgctctttcggggtgcagtctcttctgttgttcattaagatgtatggatgcactgttgtgaaggatttgtgaactggggatcgacaccccgtgaggggtgccccagtgtccataggagtttgctggagaggtgtgttgctgtagtgactatccgtgacctggcattctaaggtgttgaccccaacctgtgagggtctggatcgcagtgttgaagtgctttggagggttcaatggggtttctgtagtggatattatgtgcttgacgactactggtacgagtgtattgggggtctacatgtgtga 458 Fipivirusctcttccgatcttgggggttcgcccccatgtctcatttcaactagccgtgtgtctagttaacgcaccgc Ectcaccctggtcgttatcgggtcggttcttgcgaccgttagatcgtgagcgtttctgaggatcagttcgtataagttctccggtgtggcgaccgtaaaatcgtcacgtcccatgcaatagatgacgttaaactcgtttgccagttacataaaggaatgttgttacttttaaattgtctgttacatttaacatcttgccagtatgatgctactgtacactacgggtgtaggaaccttgtagtgtgacgtatcactcatatgtggatgggtgctccagacctttatggaagctctcagttagtagtgatccttgacttcattgagccctggtaacagtggaagtcaagatgtatatgttgctcaacacacttcggtgctacgaagctgtttgtggaagtactggcgaggttcattctgaatcatatgtttgtcacatagtcagggagtgccgtcgcttacgacggaccctttttctttataattacaaatctgtgtctcaagtgttgttggctggttttcttcttctgttttcattgttcatatatatacgtcagagtgaaagactcggtatatacaaaactgatccaga 459 Aquamavirusttcaaaggtggcgggagagttggcctcacgctgtttagcgtgagagctggctctcctgccccttcccctgagccggggatcttggctcattcccctcttttctatcctccctcattggactttacggatgacccggcataaacttgacaaccgatgttggatttcccttgtggctgtgatggaggacataccctcgggtgtagttgtgtgcgtgtcgctctgcgactcgagcttcaaagtggtgctgaaatattgcaagcgtcgttgctcgattaacggagtggtacaatcctatgaacccaagtgcattcatgcgaaagccccggaggggtgagtagcatggactcgaatcagaagagctggagctcgcttggtacggcacgtagcattgctttgcctaaagaccaagggggtatggctataggtgggggcctatagcttgtccagtgctggttgacagactcgtgctacgcgtctggttcgagtataagtagctgcaactcact 460 AvisivirusttcactcgctttccccccctctctataggggcggtcttttaattcttattaatttcctactttactatcaaaAtttcttctaagtagggactgaggtcacttagccctccctctcctgggctttccagggttatagaggttctaaagctaagccatgtgtcttgagctacacttagtacaaaggtttagtaatgattgtacatgccagtaaccttctagtgcccatggattaaagagtggtaacactctccatggggcccgaaaggctagtgggcatagttggcatcaaggaaggggtccccaccccaacctgaattgctggctagaagctcaccttagaagaagtgctgggtgacaacgtgtccaatcgtgaacgactgatggaaacgtgtggagatggatatgtgggggttcactgagtagatgccctgaaggagaaatctgatcaggggcccgtgactatacgctaggtaaaccgggtataaaaaccatgaaaggtggcccaaaatcttttccttttattttatttctatgttggtgacagtcaag 461 Avisiviruscaccccctactgccctaacccccaaagttagttatagggtggctccctacccttactccacggggta Bagccctaacccggttgaatctcaagatcagccttagcgaggactattagtaccgctcaaaccctttgcctgtagtgcccaggggtcacagaggggtgaccctctccctggggcccaaaaggctaggtggcaagacagggtccaagtgaggggctactctaagtagccccaagctgaacatcctgtctgaagccacccttgcagggccaggtttgattggggaaactagacaccagctttgtcctgggattggggggatatcgagttagtccaggaggtgcgagtagatgcccccgaaggtaccccaggcacatctgggatctgatcgggggcccgtgactatacaataggtaaaccgggttaaaaaacatgaaagcgcctctctctttcctacttcttttattgactggtgacaaaaatagcagt 462 Crohivirusgttgaagtccatttcttgcttgcccccgatgaatcctgttaaggcctcacggccctaagggtgaaact Acggttatcccctcctgtacttcgagaagattagtacaacactatgaaatctacatcttgtgatccgggataaccccaatcccagaaacctgtgatgggcgtcaccacccctcttatggtaacataagggtgtcgccgcgttggcacaggaccctttgggctggatgtttttagtaatggtgtcgaaggtcctattgagctacaggagtttcctccgccctggtgaatgcggctaatcttatccctgagcctaaggttgcgatccagcaacttgatggtcgtaatgcgtaagttgggggcggaaccgactactttccagaaggcgtgtttctttgttttgtctgttactatggtgcatgatatagatattgaatatttgatctttttgagctgtttcttatcttattgctacatcctttcaggtgttggatttacattttggttaataag 463 Kunsagivirusgattttctggttatcccttttggacttggtaggggcccacgtgcccacccacctctgtgtgtgttgattt Bctaatcgatgcctggcagtggcggccacctctccttactggtaaacctccggtgagtgaagttgtcaagctacaggtaccgtgcaggatgaaatgcgcacatgtgaacaaactaggagtcatacaccgggtcaaactctggaaacggagtccgggactctgaccaggagggtgagctcgaggcatcacattgatggacgcgattcgctatccttccctagtaggaccttgtggtgtacccctggttgggaatccagggctggtcgggtgcagggtgacagcctgttctccacctcaaccattgtaggagaaatcaacccct 464Limnipivirusttctttggatatccatttaacgtgtaccctatacgataattggggtggattctggatgcctagttccagt Agattggttaagaactcgtttactacgtatagtatgattagcaaagtgctcgattgatcacgtaatgatctatgtggttaaaaacccagtagtatggtatatactcagtagtgtacactgtgagtacaactcttggcgtagagagaacaattcacccgaatccgtggcgtatccatggaaataagtttacctaattgtatgttacaaggcatatgagacatttatgagatatggtttattttgactaaacgagtgtagaggtggtggagtctatccaacttcaagccatgcaattgttgtgttgattgatatcattgaccatttttgtggattgtgtacacatacaatttgaaaattaaccccctcaagaataagacatgggaccattcgtggtagataccgtgctcggatgcttgagattagatgggttagactagttttggaatgagattgccgagaaagtcccgctagacatgttttacaagtcgtggtattccgctagactttttcgcagacacatggaagggtccatgtgttgtgcaattgcagggtgacagcccaactgcagagttttccttactagaataaaaatctgttgtcaatttt 465 Limnipivirusgtttctgagcactggtaagagcttagacaaacgtttttaaaatttattttctctgcaacttttgtttgtg Ctttatttttatttgttaattttgcgcctaagcatttgttgcgaagtatttgattcattagtaatattacttattgtttatttagatggtattcaaagtggtgggagtatcgaacccaagcgtcgtatgctatctccttgaacaatttttaatcattgcgaagtgatcattgaaaaggataggtgtttaagaactcaaagagtgttaataatgttgggtgacaggtgtccccatagaatttattaacatgtttaggactggttatctagtaagaagaaccatcgaacgcacgagcgagcattgcttgcggggcagttaccctgcgtcgatgtaagtgtgtaccggggggtgcacatgttgattctttatggcctgatagggtgcgtcattcgcgcctagataattagtataatgcgaatggaataaatttac 466 Orivirusggtcccaggccaatattcttcgtaaggcttggttccaattttccaccactcgtgtttgggttctggcctatggtacccagaggggcggtttgggggaattaactccccctcccctgtggtcctataccaccccacacctctgtgggctttctttactatcttcttgttttccgacttttaaacactaggcaggcgcgcctagtcatacaccgcccggctggtctttccagcttttgtgggcggtgcgcgctggtccatcgtgcccagcgacatagcaccttgtggacacctccgaacgccctcccctgtatggggtggtgcccaggggtttcagtgtggtgacacactccctggggcccgaaaggctagtgtgcaacaggtgaggtacagccagctgcccccgtggctggagggaccaagcttgtgaagcacacctcaccacttgggggtgggctagtaagtggtgaaagcatagtgtccgtgtcgctggccaacactttgggtcaagtccagccactcagtgagtagatgcccaggaggtacccctagtggatctgacttggggcctgttacttaatgcaggttaaaaactatgaaagctgagtagtgtagcccggctggtggcttctcttccttattcattctattttatggtgacaaacgcaactgaagcc 467 HAV FH1cttgatacctcaccgccgtttgcctaggctataggctaaatttccctttccctgtcctttccctatttccttttgttttgtttgtaaatattaattcctgcaggttcagggttctttaatctgtttctctataagaacactcaattttcacgctttctgtctcctttcttccagggctctccccttgccctaggctctggccgttgcgcccggcggggtcaactccatgattagcatggagctgtaggagtctaaattggggacgcagatgtttgggacgtcgccttgcagtgttaacttggctttcatgaacctctttgatcttccacaaggggtaggctacgggtgaaacctcttaggctaatacttctatgaagagatgccttggatagggtaacagcggcggatattggtgagttgttaagacaaaaaccattcaacgccgaaggactggctctcatccagtggatgcattgagggaattgattgtcagggctgtctctaggtttaatctcagacctctctgtgcttagggcaaacactatttggccttaaatgggatcctgtgagagggggtccctccattgacagctggactgactttggggccttatgtggtgtttgcctctgaggtactcaggggcatttaggtttttcctcattcttaaacaata 468 HAV HM175cgccgtttgcctaggctataggctaaattttccctttcccttttccctttcctattccctttgcttgcttgtaaatattgatttgtaaatattgattcctgcaggttcagggttcttaaatctgtttctctataagaacactcatttcacgctttctgtcttctttcttccagggctctccccttgccctaggctctggccgttgcgcccggcggggtcaactccatgattagcatggagctgtaggagtctaaattggggacacagatgtttggaacgtcaccttgcagtgttaacttggctttcatgaatctctttgatcttccacaaggggtaggctacgggtgaaacctcttaggctaatacttctatgaagagatgccttggatagggtaacagcggcggatattggtgagttgttaagacaaaaaccattcaacgccggaggactgactctcatccagtggatgcattgagtggattgactgtcggggctgtctttaggcttaattccagacctctctgtgcttggggcaaacatcatttggccttaaatgggattctgtgagaggggatccctccattgccagctggactgttctttggggccttatgtggtgtttgccgctgaggtactcaggggcatttaggtttttcctcattcttaaataata 469 Parechovirusggtcggggagatgtgttcatgatcggttaacaccatcatggatcatctctccccgacctctttttgacc Fcagctatgggttaaatagtacttttcttttctcttttgctttcttttgtgtttgtttgattgcaacatataacaagcattttatcagtattagtgtctgcaactgtataacaagcaaggtggagcaatcatgcgagtatatctcaattgaattgtgacacacaagtgtgcactatgtggaataaatgccattttggccaaacctggttagccagaccagtagtaggacaatttggcacccttagtgggcgcgacctagatgctagggatgagcaaacctatttcccctgagtacaggggctctccttcacctctacttttaggacctctttttgagtatcctcgatagaaggtgaagtgacggtgtaccggatggttaattgatctcattgctgggtgacagcccgctaggaccaggcagcatctttgtatggacctgtacatgtaac 470 Parechovirusacatggggcaggtgtgctgtgccaagagcaacactacggtggccgagccgatggttcgtcacca Dcgtagtaggactccgtagtgcttggttacggcggacgtaagtcagttgagtgatgtctaagtggcaaaccatgagtacatggtaaccttgtgtggactcgcgggacggaatttcctatcccattgactccttgtagcaaggtgggtatacccaaccacaatggcagcaccctgggtgggaacccaggggcctggattagtatccagtcacacagcctgatagggtggcggctcagccactgaccagcgtctctaaataattgtgagctgttcatgcacc 471 Parechoviruscggtcatccccctttccccacagccggtgtgggttctaatcggctcctactaaacacctaagcatca Cctgcgcctctatctctcctatccacaggtctaagacgcttggaataagacatgtgggtgcaataggaagattagctagtccaatctctccttccagctacgcttctcccttcgatgagcgtagggggggcccccacctccctcatctctggatagggctcttgctacggggctttcccgtctggaccagcaggcccactggtgcgcttccattcaagtttagtgtgcattactgtctgaaatattgctttgctaggatctagtgtagcgacctgcatattgccagcggacttccccacatggtaacatgtgcctctgggcccaaaaggcatgtctttgaccgtatgcagtacaaccccagtataggtcctttctatggcagtatggatctcagtgatgagtctatacagaatatggaagtggttcggatatgtcagcccgaaggatgcccagaaggtacccgcagataaccttaagagactgtggatctgatctggggcccaccaccttcgggtgggtagaagctaaccatgccttgggttaaaaaacgtctaagggctgaccagacccgggggatccgggttttccctatcttgacctactctaatc 472 LjunganctcattgcccacacctggttggttcccaggttcatacaataaccatcaataaacttttaacatctaagatVirusagtattatcccatactagactggacgaagccgcttggaataagtctagtcttatcttgtatgtgtcctg87-012cactgaacttgtttctgtctctggagtgctctacacttcagtaggggctgtacccgggcggtcccactcttcacaggaatctgcacaggtggctttcacctctggacagtgcattccacacccgctccacggtagaagatgatgtgtgtctttgcttgtgaaaagcttgtgaaaatcgtgtgtaggcgtagcggctacttgagtgccagcggattacccctagtggtaacactagcctctgggcccaaaaggcatgtcatttgaccactcaggtacacaaccccagtgatgcacacgcttagtaatggcttagtaacaaacattgattgatcatttgaaagctgttaggaggtttaggtatgacgggctgaaggatgccctgaaggtacccataggtaaccttaagcgactatggatctgatcaggggcccaccatgtaacacatgggtagaagtcttcggaccttgggttaaaaaacgtctaggcccgccccccacagggatgtggggtttcccttataaccccaatattgtat a473 Parechovirusgccgtcgggccttacaccccgacttgctgagtttctctaggagagtccctttcccagccagaggtg A2gctggtcaaacaataccaaacgtaactaaacatctaagataacatagccctatgcctggtctccaccagttgaaggcatcttgcaataaaatgggtggattaagacgcttaaagcatggagtcaattatcttttctaactagtgatcttcactgggtggcagatggcgtgccataactctattagtgggataccacgctcgtggatcttatgcccacacagccatcctctagtaagtttgcaaggtgtctgatgaggcgtgggaacttattggaaataattacttgctgcgaagcatcctactgccagcggatcaacacctggtaacaggtgcccctggggccaaaagccacggtttaacagaccctttaggattggttaaaacctgagtaattatggaagatacttagtacctaccaacttggtaacagtgcaaacactagttgtaaggcccacgaaggatgcccagaaggtacccgcaggtaacaagagacactgtggatctgatctggggccacctacctctatcctggtgaggtggttaaaaaacgtctagtgggccaaacccaggggggatccctggtttccttattttagtgtaaatgtcatt 474 Parechovirusagagtccttttcccagccagaggtggctggttaaataatacctactgtaacaaaacatctaagatgta A3acaaccacacacctggtctccactggccgaaggcaactagcaataaggcaggtgggttcagacgcttaaagtgtgttgtacatattcttttctaacctgtgttttacacagggtggcagatggcgtgccataactctaacagtgagataccacgcttgtggaccttatgctcacacagccatcctctagtaagtttgtaagatgtctgatgacgtgtgggaacctgttggagataacagtttgctgcaaagcatcccactgccagcggatctacatctggtaacagatgcctctggggccaaaagccaaggtttaacagaccctttgggattggttcaaacctgaactgttatggaagacatttagtacctgctgatttggtagtaatgcaaacactagttgtaaggcccacgaaggatgcccagaaggtacccgtaggtaacaagtggcactatggatctgatctggggccagctacctctatcttggtgagttggttaaaaaacgtctagtgggccaaacccaggggggatcctggtttctttttaatttaagtaatcact 475 Parechovirusgggccttataccccgacttgctgagtttctctaggagagtccctttcccagccctgaggcggctgga A8taataaaggcctcacatgtaacaaacatctaagacaaaataatttgccttgcacctggtccccactagttgaaggcatctagcaataagatgagtggaacaaggacgcttaaagtgcaatgatagttatcttttctaacccactatttatagtggggtggtggatggcgcaccataattctaatagtgagataccacgcttgtggaccttatgctcacacagccatcctctagtaagtttgtgagacgtctggtgacgtgtgggaacttactggaaacaatgctttgccgtaaggctttcattagccagcggaccaccacctggtaacaggtgcctctggggccaaaagccaaggtttaatagaccctaatggaatggttcaaacctggagcattgtggaaagtacttagtacctgctgatctggtagtaatgcaaacactagttgtacggcccacgaaggatgcccagaaggtacccgtaggtaacaagtgacactatggatctgatctggggccaactacctctatcttggtgagaggttaaaaaacgtctagtgggccaaacccaggggggatccctggtttccttttattttactttgtcaa t476 ParechovirusctctattagtgagataccacgcttgtggaccttatgctcacacagccatcctctagtaagtttgtaagaA17 cgtctggtgacgtgtgggaacttgtgggaatcaatattttgctttaaagcatccattagccagcggataaaacacctggtaacaggtgcctctggggccaaaagccaaggtttaacagaccctagtggattggtttcaaaacctgaaatattgtggaacacactcagtacctactgatctggtagtaatgcaagcactagttgtaaggcccacgaaggatgcccagaaggtacctgtagggaacaagagacactatagatctgatctggggctggctacctctattttggtgagtcagttaaaaaacgtctagtgggccaaacccaggggggaccctggtttccatttattttacaaaggcact 477 Potamipiviruscacatggaaagcttttcgcttccatgtttacgcacacactctctttgacaccctgttgtatggtgttaaa Actacaacatttgtctgtctataatcgtttattttgtttaccctatatgtacccaagtatttgattgcttgactcacataagcatcggtaacccatactgttttatgagctactacctctgctgtctacatacattttatatgaatggtttgagctctgcctcaggatcaaacatggtaacatgttcctttggtcagttagaatcttattgtataatctaaggtgtctattagtacgtagaaagttgtaacacatatggggcctgatagccgctatctctgatggatgtaaggtaaccttctttaggtctgatacattctgcacaggatccaattttcggtgccctgtacgagtgcactcttatgcacgaggacgagatatgctacaacccactgcaaatttaaacccaaactttaaca 478PotamipivirustttcaacgtcgtggctgacgttaaaaagccacaattccacttaccttttaccttttatgtttaatgtttgttaBgttttgtgatctttaacaaatagatctaaataatttgttggtaaccaatctcggatgtttcggctgcattgtagtttatttatttcattttagttgtaggtggccactacgtcctggaatcatacatggtaacatgtacctcggcggttatccactattacgctaatctaagaatatttaaatgaaaatgtaagtgttacggctgactttgggcctgatagttaaatgctcgcactgacagatagtaccctcctttaggatcgattctgttacatgggatccattttggtgccccactgattcaacctctttgagaaaaagagttagcatactacaaattttccaaacaaaaaccctttttaatgactacaacttatgatattttgaattttactgctcttgaaaaagatattttgacattgatcgctgtactgtttcagacattcattgcatccatttttgttggctactcctcacaaactcaaaacttttccacacgagaaaccttgtttattgaattttgcctttatttttaggaacttgttgttggatttattgtttgcttaattattgacctcacacctgttttaaacactacaat 479 BeihaigggacaaccccacagctggtacaaccattgtgggttggtctccaccctttttcaaccgtggcaacttCongercggttaaagttgcaaatcccccctctccctattccacctcccttactacactccccatatatggtcccaPicornavirusgattttattctacctctttatatttttatttagtacagtggtggtgaattactcccagcataaactttgctggatcagtgttcatcaagcatactaattactaatgtactgagctatactattatctggcatctcacctggataaccggtgtgaccatatttcctaggttgcctccctatgtattttgtagcacctgtgcatctgcacgttggggcgacaaattgtaggtttcctggcacgggtaagaattgtggaaagctagtatgcagttaatgcaagggcgcgtttttcgctaccccgacactgctaaagtttttgggaggggtcccttaaacatttctagtattgagtgatagctttgcggcaggtcaccacaaccttactataaataaacctgttgaatctcac 480PorcinetacgcatgtattccacactcatttcccccctccacccttaaggtggttgtatccccataccttaccctccSapeloviruscttccacaatggacggacaaatggatttgacctcacggcaaacacatatggtatgatttcggatacaJD2011ccttaacggcagtagcgtggcgagctatggaaaaatcgcaattgtcgatagccatgttagtgacgcgcttcggcgtgctcctttggtgattcggcgactggttacaggagagtaggcagtgagctatgggcaaacctctacagtattacttagagggaatgtgcaattgagacttgacgagcgtctcctcggagatgtggcgcatgctcttggcattaccatagtgagcttccaggttgggaaacctggactgggcctatactacctgatagggtcgcggctggccgcctgtaactagtatagtcagttgaaaccccccc 481 PorcinettgaaatgggtgtggggtacatgcgtattacggtacgcatatattccacactcatttccccccctccaSapeloviruscccttaaggtggttgtatccccataccttaccctcccttctaaaacagatggacaaatggatttgaact A2tatggcaagtgaatatggtatgactttggatacactttaacggcagtagcgtggcgagctatggaaaaatcgcaattgtcgatagccatgttagtgacgcgcttcggcgtgctcctttggtgattcggcgactggttacaggagagtaggcagtgagctatgggcaaacctctacagtattacttagagggaatgtgcaattgagacttgacgagcgtctcttagagatgtggcgcatgctcttggcattaccatagtgagcttccaggttgggaaacctggactgggcctatactacctgatagggtcgcggctggccgcctgtaactagtatagtcagttgaaacccccc 482 SimianccaaggatctgttgcataggcgttgtatcccctaaccttttacctacccatcccaataggactggtattSapelovirustcggttttgattgagtaatggatactgattctatacctgttacccattcaggggaaaaatggagtttcttt1 catggatctgacttgatatgaccaagagtcaacactttgcgtgttggccgtatggaatgctttaaggtttattctttggattatgacttcagggttggccgcccaggataaaaggcaattgtggtaagtgatgttagtcattggtggttgaaacctgcctaagacgtcctaggtctacgctgtgcgggccgaagtaagcttaggaataacagggagtatgccattttctgctttcacccaacacgaccgtacacgaaagagctagaggcactttggggcaaagggaaaagctttgcttagcccgaatgttcatttgagtccttgacgaatgcgtcccgtctgtcccgacggtgaggcgtatggcgcatgctcatggcattacccaatggtgtatctgtgaggggggggctcctcacacttagtctagtgctacctgacagggccgcggctggtcgtttgtgtatggtataaccagtagtaatcccccatggattgctttaacttcccctcctcccttaccaagacattctctaag 483SimianttttaacttgttatgacattcaaggaaaaaatgtctttttcattatgggactgacctgtttatgaacatgagSapeloviruscagcggcactgctccacgggctatccgtgtaagaaatattgattattcttatggatcatgatttcagg 2gttggccgcccagtctaaaaggcaattgtggtaagctatgtaagtagttggctgttgaaaggagccaagtacatcctaggtctacgctgtgcgggccgaagtaagacttggaacaactctgagtaggcagtttttctctttagcccaacacgaccgcatactgaagagctagaggcactttggggcaaaggtaaaagcattgcttagaccgaatgttcaatgagaccttgacgagtgctgtcacagtgtcccctgatggcagtatggcgcatgctcttggcattacccatatgtgtatctatagggggggggccccctatacttagtctagtgctacctgacagggccgcggctggtcgtcggtgtgtggtataaccagtagtaatcccccatggattgctttaactccccctcctccctcaacaaaactttctctaag 484 Rabovirusccgggtataacccggagttttggggcaggtccaagccccacataggaacatacgatccacggatc Cgtgtgttcttttatgctttctaaccttaccctttgtaaccattacgctttacgccgcatggtgtttggcggcaccatgacgtggacaagaggttacgccattacgatatgtaccctccctttaggggagagaccgaccaattatggtacagtatccaactgtattgtggtcaagtttcactgtttccccggtgatgcgggataggctgtacccacggccaaaacctgctgatccgttacccgactcacatctacgaggaggctagtaaaaggcatgaagttcaagagtatgatccaaccagatccccactggtaaactagtgatgagggttcccgaccgaacatggcaacatgtgggttccctgcgttggcactaggccccttccgaggggtgctctgaagatggattgttgatgaagaccaatttgtgcatgtgtttatcctccggccctctga 485 Rabovirusccgaccccactggtcgaaggccacttggcaataagactggtggaacaaggtcgcctgtagttgatt AggaaccttctttctaatgacttatgtcagcggtgctactcacaccgtaactctcctaccctatccccacNYC-B10gcttgtggaactaggaggggatgagtgattcaagtaagtactgtcagaatggtgaaaataatctgattctgaaacgctatggatccatcgaaagatggggctacacgcctgcggaacaacacatggtaacatgtgccccaggggccgaaagccacggtgataggatcacccgtgtagtttgagatcatatcaatgttcatagtctagtaagatgatttgaaatctaactggtctgatggctaactgcttgtcttattgcggcctaaggatgtcctgcaggtacctttagagaaccattagagactattgatctgagcaggagccaaggtggtctttcccagccttggttaaaaagcgtctaagccgcggcagggggcgggaggccccctttcctcccaaactataatatagattgt 486 Parabovirusgatgtatccccatcccccagtgtgtatgccatactgcatagctcgcctatgccctatggattcacaac Ccctttcatataccctccctacccaaccccgtaaccacatgctttactccgcttggggttttgcggccccatgttgtgacgaaatggctacgcaatcaatgcggctaatggggcctgccgcttttaagtggccccagttagaagtttatgcacacccgcccattaggaggccaccagccaggtggtcagagggcaagcacttctgtttccccggtgaagtttgataagctgtgcccacggctgaagcagacagatccgttacccgcctcactactacgagacggctagtagtgtgtaatatccgaatttcattgatccgggtgttccccccacccagaaacgtgtgatgaggagcggcacccctcctatggcaacatagggcctctcctgcgctggcacacgggctctatgagcatgaaatcaggagaaagtcacacgaagaccaattgtgctagtgagattcctccgcccccctgaatgcggctaatcccaactccggagcgcccgctggcaaacccgccagaagagcgtcgtaatgcgtaagtctggagcggaaccgactactttgggtgtggcgtgtttcctttatttcctttgtatttgtat 487 Parabovirusaacccataatccattgtccatcaatgttttatgggggggaccctttctcccctccccctccaaatacct Btttacccctctgtaaccaagagtgtgcaaaatctatttactagcccagaattgcggcttctggggaggtttattcctcatgcctaacaagatgttacgcaaactccgggctacggccctgggcttttgccctaaagatttagaagtttacactatcgtccaacaggaggacaacaaaccagttgttctaaggacaagcacactgtttccccggtgagactggatagactgtacccacggttgaaactggttgatccgttacccgactcactacttcgagaagattagtaggaaactgtgaaactgattccattgatccggatactttccccgtatccagaaactactgatgagggttgacttcccgactacggcgacgtagtgtcatccctgcgctggcagtaggcctctttgaggatggaagatgtggatcggtaaccgaaggtcctattgagctagtgtttatacctccggcctcctgaatgcggctaatcctaacccatgatctagtgctcacaaaccagtgagtagctagtcgtaacgcgtaagtcgtgggcggaaccgactactttggagtgaccgtgtttcctattttacttttgtttg 488Parabovirusaccgttacgcaccactcagttggtgtttggtggcaccaatgatggaacaaaaggctacaccacttg A3ggctacggcccgcgccaccttgtggcgcaaagacattagaagaatagcataccgcccactagggccctgcagccagcagggtaacgggcaagcacttctgtctccccggtagaacggtataggctgtacccacggccgaaaactgaactatcgttacccgactccgtacttcgcaaagcttagtaggaaactggaaagttcgagttattgacccggagtgttccccccactccagaaacgcgtgatgagggttgccaccccgaccatggcgacatggtgggcatccctgcgctggcacgcggcctctaagaggataactcgctcctactggtaaccgaagagccccgtgagctacggtttattcctccgcctccctgaatgcggctaatcctaacccatgagcagttgccatagatccatatggtggactgtcgtaacgcgtaagttgtgggcggaaccgactactttgggatggcgtgtttccttgttttctccatttgttgttgtatggtgacaagttatagatctcgatctatagcgtttcttgagagtttaccaaacatttattcaagtcgtacaattcttgtgtttaagcagtacagtgtaagg 489 Felipivirusgatgtcggatgacggctggccaccggggaaaaacggcaaatgtgcaccacctctgcaacccac 127Fgccgaccacgtttaaccatggcgttagtaggagtggaccactgcagtgggctctggtgtgcgacagtcagtggtagagtagacagtcctgactgggcaatgggaccgcgttgcgtatccctaggtggcatcgagattcctctgctacccaccagcgtggactcctatggggggggccccataggctaggtctatactgcctgatagggtcgcggctggtcgaccactgactgtataaccagttgtaactcact 490Boosepivirusttgaaagacctcggcatatatcgttgtcacaacggtatatgtcgagatctttctccccaccccctcca Aattcccttttccccctcttgcaacttagaagtagtttcacacagggcaataggatacgtgatccagccaggacacgtgagctcaagcacttctgtttccccgtccccttcacgtactacgggaatgttagtaatttgtgtgcactttagtaaggttgatccgggattaaccccaaatcccagaaactggtgatgagcgttaccacccccgccgggcgaccggaaggtttcgctgcgaggcaccagggcttcggcaccagaaaaaggtaaagcaaatgaaggcgctactgtgctacgagaagtttcctccaggcccctgaatgcggctaatcctaaccagtgatccaccggtgcaaaaccatgtactaggtggtcgtaacgcgcaagtcgctggcggaaccgactactttgggtgtcctgtgtttccatattttattttattcaattttatggtgacaagagtaaagagatacagatttgcagcc 491 Boosepivirusttttctcccctccccctccaactaccctttccccctcttgtaacgctagaagtttgtgcaaaccgcctgt Bagggtactgcaatccagcagtgcataggctaagcttttcttgttaccccaccccacattatactgaggaggattgtgaaattgtgttagtatgggttagtagcggtgacccgggtaaccccaacccagaaactcacggatgagatgaacaggaccccacatggtaacgtgtgtgttcgtctgccccgcaaggtgaggccgtgagagctttgcacgcgaaaaccttgaaaacccaaaagtaccttgagctcttcgctattttgtgtttcctccaggaccctgaatgcggctaaacctaacccgcgatccgcacgtagcaacccagctagagtgtggtcgtaatgcgcaagttgcgggcggtaccgactactttggtgttcctgtgtttcctttattttattttgaatttttatggtgacaacagctagaaaataagagtgaac 492 PhacovirusgtgtgtcatttctcccctccccctcccaaaccttttccccctctaatcggattgattaacccggttaaagPf-CHK1atgattaatggtttgtgagttgatatgatggcccggcattgaatccgggaattcttaagtaatggaattgcatccaatatgaaagtgagtgtggcaagctcacaagtagtacttggctctgcccattatttgaggacaactcttcttgactacaatgtgtttaaagtaaactggaccacattgtgtatccagacaactccatttgataatgtacgctggaaacgttttcagtgcatagggtcctaaagtggtgctgaaatattgcaagctcaatgggatactgaacgctgaaaaccgccgctgttatcatatgggcccctagtgggtaaatgttggctttaggcatatactgcttgggaatgcagtactggttgtagacagggtgatagcctaccggctggcgtagttgagaggtatagccagttgattgccat 493 HRVC3ttaaagctggatcatggttgttcccaccatgattacccacgcggtgcagtggtcttgtattacggtac QPMatttccataccagttttatacaccccaccccgaaactcatagaagtttgtacacaatgaccaataggtggtggccatccaggtcgctaatggtcaagcacttctgtttccccggcacccttgtatacgcttcacccgaggcgaaaaatgaggttgtcgttatccgcaaagtgcctacgaaaagcctagtaacactttgaaaacccatggttggtcgctcagctgatacccaacagtagacctggcagatgaggctagacattccccaccagcgatggtggtctagcctgcgtggctgcctgcacaccctgccgggtgtgaagccagaaagtggacaaggtgtgaagagcctattgtgctcactttgagtcctccggcccctgaatgtggctaaccctaaccccgtagctgttgcatgtaacccaacatgtatgcagtcgtaatgggcaactatgggatgggaccaactactttgggtgtccgtgtttcctgttttactttttcattgcttatggtgacaattgtatctgatacacttgttacc 494 HRVB27ttaaaacagcggatgggtatcccaccatccgacccacagggtgtagtgctctggtattttgtacctttgcacgcctgtttccccattgtacccctccttaaatttcctccccaagtaacgttagaagtttaaggaaacaaatgtacaataggaagcatcacatccagtggtgttatgtacaagcacttctgtttccccggagcgaggtataagtggtacccaccgccgaaagcctttaaccgttatccgccaatcaactacgtaatggctagtattaccatgtttgtgacttggtgttcgatcaggtggaccccccactagtttggtcgatgaggctaggaactccccacgggtgaccgtgtcctagcctgcgtggcggccaacccagcttttgctgggacgcctttttacagacatggtgtgaagacctgcatgtgcttgattgtgagtcctccggcccctgaatgcggctaaccttaaccccggagccttgcaacataatccaatgttgttgaggtcgtaatgagtaattctgggatgggaccgactactttgggtgtccgtgtttccttttattctttatattgtcttatggtcacagcatatatagcatatatactgtgatc 495 HRVA73ttaaaactgggtttgggttgttcccacccaaaccacccacgcggtgttgtacactgttattccggtaaccttgtacgccagttttatatcccttcccccccttgtaacttagaagacatgcgaatcgaccaatagcaggcaatcaaccagattgtcaccggtcaagcacttctgtttccccggctctcgttgatatgctccaacagggcaaaaacaattggagtcgttacccgcaagatgcctacgcaaaacctagtagcatcttcgaagatttttggttggtcgctcagttgctaccccagcaatagacctggcagatgaggctagaaataccccactggtgacagtgttctagcctgcgtggctgcctgcacacccacacgggtgtgaagccaaagattggacaaggtgtgaagagtcacgtgtgctcatcttgagtcctccggcccctgaatgcggctaaccttaaccccgtagccattgctcgcaatccagcgagtatatggtcgtaatgagtaattacgggatgggaccgactactttgggtgtccgtgtttcactttttacttatcaatttgcttatggtgacaatatatatagatatatattgacacc 496 EV Lacatgggccagcccaccacacccactgggtgtagtagtctggttctatggaacctttctacgcctcttttgcttccctcccccatttctccttcgattgctccacctgtgatctttgcaacttagaagaaataatgaacccgcacaatagcgggcgctgagccacagcgtcaatgtgcaagcacttctgtttccccggaatgggcccataggctgtacccacggctgaaagggaccggcccgttacccgccttggtactgcgagaatgttagtaactccctcgatagctttaggcgttacgctcagccctttgagcccgaagggtagttcgggtcgatgaggctcgtcattccccactggcgacagtgtgacttgcctgcgttggcggcccggggtggggggcaacccccatccacgcctactgaaggacagggtgtgaaggcgctattgcgctactaaggagtcctccggcccctgaatgcggctaacccgaaccccgagcccacggtggtaaacccgccacaagtgggtcgtaatgagtaatttggggcagggaccgactactttgggtgtccgtgtttcctgtttttccatacgatggctgcttatggtgacaaccataagcaattggattggccatccggtgttcatattgcgaat 497 EV Ktcagcctgacgcaagtgcctccattggagtctctccaagccctccggggcttggagggcgccgaccccctgcctagggggagcccacgacacggctggagtccattggcacaccgcagccacgattcaagccagaattgaaagcgggaagcacttctgtctccccggtgtggatcatacgctgtacccacggcgaaaagtgaagcatcgttacccgactcggtacttcgagaagcccagtacagttgtggatctctgcagggtatacgctcagcgtgacccctacgtagttccttgagatggctgagagaacaccccacgggcgaccgtgtctctcggcgcgtggctcaaggccgggccttcagtggctcggtgccttgcagagtgaagcctccgaacagcctattgagctaccgtttagcctccgccctcttgaatgcggctaatcctaaccatggagcgcccgcccacagtccagtgggtagagcgtcgtaacgcgcaagtccgtggcggaaccgactactttagagtggcgtgtttccaatttatcctttataaagttgcttatggtgacaccacaagagatccacgatttcttgtttcttatcactgagacacaagtcatattcatcaatctttattgcggaattaacttggtgcgtccaaacacatcagc 498 EV J 1631caccctgagggcccacgtggcgtagtactctggtatcaaggtacctttgtacgcctattttatttcccttcccccacagtaacttagaagcttatctcatagttcaacagtagggtcactaaccaagtggctcagcgaacaagcacttctgtttccccggtcctagtacctgtgaagctgtacccacggcggaaggggaaaaagatcgttatccggccccctacttcggaaagcctagtaacaccattgaagcaatcgagtgttgcgctcagcacagtaacccctgtgtagctttggttgatgagtctgggcactccccactggcgacagcggcccaggctgcgttggcggccaaccgactcgggcaaccgggtcggacgctcgtttgtggacatggtgtgaagagcctactgagctagagggtagtcctccggcccctgaatgcggataatcctaaccccggagcacccacactcaatccagagtgcaggatgtcgtaacgcgtaagtctgggacggaaccgactactttgggtgtccgtgtttcctgttttacttactttggctgcttatggtgacaatctagtgttgttaccatatagctattggattggccatccggtgttttgaattgtgtgtttatactaattcttttacatatcacagacaaccaaat 499 EV J N125cggtacctttgtacgcctattttacccccttccccttgtaacttagaagcaaagcaaaccagttcaatagtaagcaacacaacccagtgttgtgacgaacaagtacttctgtttccccgggagggtctgacggtaagctgtacccacggctgaagtatgacctaccgttaaccggctacctacttcgagaagtctagtaataccattgaagttttgttggcgttacgctcaacacactaccccgtgtgtagttttggctgatgagtcacggcattccccacgggcgaccgtggccgtggctgcgttgcggccaaccaaggggcgcaagctccttggacgtcacttaacagacatggtgtgaagaacctattgagctaggtagtagtcctccggcccctgaatgcggctaatcctaactccggagcacatcagtgcaacccagcatttggtgtgttgtaatacgcaagtctggagcggaaccgactactttgggtgtccgtgtttcctgttttaccttatttggctgcttatggtgacaatttgatattgttaccatatagctgttggattggccatccggatttttgaaagagacccaaaactttcttctctacttcagattcaagtgcgaagttttccttttcatatattacttactaatttgaagtaccaaag 500EV Ittagtactttctcacggggatagtggtatccctccctagtaatttagaagacttgaaaaaccgaccaataggcacctcgcatccagcggggtaaaggtcaagcacttctgtttccccgggtcgagtagcgatagactgtgcccacggtcgaaggtgaaacaacccgttatccgactttgtacttcgggaagcctagtaccaccaaagattatgcttggggtttcgctcagcacgaccctggtgtagatcaggccgatggatcaccgcattcctcacggcgactgtggcggtggtcgcgtggcagcctgccgatggggcaacccatcggacgccaagcatatgacagggtgtgaagagcctactgagctacaaagtattcctccggcccctgaatgcggctaatcccaaccacggagcatttgctaccaaaccaggtagtggaatgtcgtaacgggtaactctgtggcggaaccgactactttgggtgtccgtgtttccttttaatttatcattctgtatatggtgacaactatagtgctatctcgatttgcattactattgttgagattaaaactttattacattgttgcattttaccctttgagtgagttttcacctgaacagattaatttactcatcctgtttatatattacaagcagaaatacttgcaaag501 EV F1gcaatgctgcaccagtgcactggtacgctagtaccttttcacggagtagatggtatcccttaccccgBEV 261 gaacctagaagattgcacacaaaccgaccaataggcgcaccgcatccagccgtgcagcggtcaagcacttctgtctccccggtctgtaaagatcgttatccgcccgacccactacgaaaagcctagtaactggccaagtgaacgcgaagttgcgctccgccacaaccccagtggtagctctggaagatggggctcgcaccacccccgtggtaacacggttgcctgcccgcgtgtgcttccgggttcggtctcgtgccgttcacttcaacttcacgcaaccagccaagagcctattgtgctgggacggttttcctccggggccgtgaatgctgctaatcccaacctccgagcgtgtgcgcacaatccagtgttgctacgtcgtaacgcgtaagttggaggcggaacagactactttcggtaccccgtgtttcctctcattttatttaatattttatggtgacaattgttgagatttgcgctcttgcaacgttgccattgaatattggcttatactatttggttgccttttacaaaacctctgatatacccagttcttacattgatctgcttgtttttctcaatttgaagtatagactacaaatagcaaa502 EV D94cgtggcggccagtactctggtatcacggtacctttgtacgcctgttttatatccccttcccccgcaacttagaagaaaacaaatcaagttcactaggagggggtacaaaccagtaccaccacgaacaagcacttctgtttccccggtgatgtcgtatagactgtaaccacggttgaaaacgattgatccgttatccgctcttgtacttcgaaaagcccagtatcaccttggaatcttcgatgcgttgcgctcagcactcaaccccagagtgtagcttaggtcgatgagtctggacactcctcaccggcgacggtggtccaggctgcgttggcggcctacctgtggtccaaagccacaggacgctagttgtgaacaaggtgtgaagagcctattgagctacaagagaatcctccggcccctgaatgcggctaatcctaaccacggagcaagggtacacaaaccagtgtatatcttgtcgtaacgcgcaagtctgtggcggaaccgactactttgggtgtccgtgtttccttttgtttttatcatggctgcttatggtgacaatctaagattgttatcatatagctgttggattggccatccggtaatttattgagatttgagcatttgcttgtttcttcaacaatttcacctattcattgcatttcagcagtcaaa503 PV3tacctttgtacgcctgttttatactccctcccccgcaacttagaagcatacaattcaagctcaataggagggggtgcaagccagcgcctccgtgggcaagcactactgtttccccggtgaggccgcatagactgttcccacggttgaaagtggccgatccgttatccgctcatgtacttcgagaagcctagtatcgctctggaatcttcgacgcgttgcgctcagcactcaaccccggagtgtagcttgggccgatgagtctggacagtccccactggcgacagtggtccaggctgcgctggcggcccacctgtggcccaaagccacgggacgctagttgtgaacagggtgtgaagagcctattgagctacatgagagtcctccggcccctgaatgcggctaatcctaaccatggagcaggcagctgcaacccagcagccagcctgtcgtaacgcgcaagtccgtggcggaaccgactactttgggtgtccgtgtttccttttattcttgaatggctgcttatggtgacaatcatagattgttatcataaagcgagttggattggccatccagtgtgaatcagattaattactcccttgtttgttggatccactcccgaaacgttttactccttaacttattgaaattgtttgaagacaggatttcagtgtcaca 504 EV C102ctttgtacgcctgttttacatcccctcccccacgtaactttagaagcaattcaacaagttcaatagagggggtacaaaccagtatcaccacgaacaagcacttctgtttccccggtgattttacataagctgtgcccacggctgaaagtgaatgatccgttacccgctcgagtacttcgaaaagcctagtatcgctttgggatcttcgacgcgttgcgctcagcactctaccccgagtgtagcttaggctgatgagtctgggcattccccatcggcgacgatggcccaggctgcgttggcggcctacccatggctaacgccatgggacgctagttgtgaacaaggtgtgaagagcctattgagctactcgagagtcctccggcccctgaatgcggctaatcccaaccacggatcaggtgcctccaacccaggaggtggcctgtcgtaacgcgcaagtctgtggcggaaccgactactttgggtgtccgtgtttccttttatcttttaaatggctgcttatggtgacaatcatagattgttatcataaagcgaattggattggccatccggtgaaatacaaacacattatttacttgtttgttggatttactccgctcacacagcttactcctaagataatatttattgtattgctggtaaggagacactattata505 EV 30aagcaaggcaaacctgaccaatagtaggtgtggcacaccagccgcattttggtcaagcacttctgtttccccggaccgagtatcaataagctgctcacgcggctgaaggagaaaccgttcgttacccgaccagctacttcgagaaacctagtaacactatgaacgttgcggagtgtttcgttcagcacttcccccgtgtagatcaggtcgatgagtcaccgcattcctcacgggtgaccgtggcggtggctgcgttggcggcctgcctacgggttcgcccgtaggacgctctaataccgacatggtgtgaagagtccattgagctagctggtagtcctccggcccctgaatgcggctaatcctaactgcggagcaggtgctcacagaccagtgagtagcctgtcgtaacgggcaactctgcagcggaaccgactactttgggtgtttttccttttttcttctcttatattggctgcttatggtgacaattaaagaattgttaccatatagctattggattggccatccggtgacgagcagagccattgtttacctctttgttggatttgtacctttgaaccacaaagtcttgaataccattcatctcattttaaagttcaactcagctaaaagaaa 506 SA5agtacttggtattccggtacctttgtacacctatttacaaaccctaccccttgtaaccttagaagcaattatttaaccgctcactagggggtgtgctatccaagcacatcaagagcaagcacttctgtctccccgggaggggctaatggtacgctgtgcccacggcggaaatgagccctaccgttaaccggcagtctacttcgggaagcccagtaactacattgaaactttgaggcgttacactcagcacataaccccaatgtgtagttctggtcgatgagccttggcatcccccacaggcgactgtggccaaggctgcgttggcggccagcctgcggaccaaaagtccgtaggacgcctaattgtggacatggtgtgaagagcctactgagctagactgtagtcctccggcccctgaatgcggctaatcctaaccctggagcatccgcgtgcaacccagtacgtagggtgtcgtaatgcgtaagtctgggatggaaccgactactttgggtgtccgtgtttcttgtttttcatactgggtcgcttatggttacaactaattgttgtaatcattggcagtgcgcgctgaccacgcgattattgatatttccatttgttggatactccaatagtgtcaactcatatacacaacttttaccactgatcaagataaaa 507 EV A114tgtgcgcctgttttgaaaccccctcccccaactcgaaacgtagaagtaatgtacactactgatcagtagcaggcgtggcgcaccagccatgtctcgatcaagcacttctgtttccccggactgagtatcaatagactgctcacgcggttgaaggtgaaaacgtccgttacccggctaactacttcgagaaacctagtagcaccatagaaactgcagagtgtttcgctcagcacttcccccgtgtagatcaggtcgatgagtcactgcaatccccacgggtgaccgtggcagtggctgcgttggcggcctgcctatggggcaacccataggacgctctaaggtggacatggtgtgaagagtctattgagctagttagtagtcctccggcccctgaatgcggctaatcctaactgtggagcgcatactcccaaaccagggagcagtgcgtcgtaacgggcaactccgcagcggaaccgactactttgggtgtccgtgtttccttttattcctatactggctgcttatggtgacaattgagagattgttaccatatagctattggattggccatccagtgtgtaatagagcaatcatttaccaatttgttggatttactccattaacccacacgtctctcaacacactacatttcatcttactactgaacactagaaa 508 MobovirustattctcccacaaaccttcttgtaactctgttaagccttttacatccatgtaatttaattttctccacctaaaA aggatttcccccatggtcctttttggctcgaacaaatgctacatagggtcttgttttctccccctggctctcttgccagggttccataccccaattcctctatttccatgatttttcatcatggtttatttttactgtcttcttattttctgaggtgaccaactcctaagccgactgggtcgcggaagcccggactcctcgcatcactagggtgcgtagcgatgtaggcgaaaatattggttgctagatgcatacatatagtgaattgatactacaccaaactctgttctttttgaaactagctattttctaagtaaggtaggctacgggtgaaaccttaccattgcaggtacgtgaaccgcaacggacatttggccgaagactggtgtacccacgtcagttataggacctcttcaacgttggtggacggcatgtcactgattagttaggctagtgaatttaagttcagggggtatcttttagcttaagcgtgtattctagtaggacttgcagagcctccccacctaggaggatctctgtttatagccccttttccttgttccgttagttttccacacttttacaatatttgatgatttgtt 509 BurpengaryctccccccccttccccttcccgagtaggagattggcatgtatgctctacatgcccgattctctcttgctViruscactctcttaaatcctggtggcggtctcggattaaacatttatgtcgtatctgggatcgtcttacttggtggtaattcctctgttgcctagggacctccggactgccggattaaaggtctcaacagagggcaatgtacaaggaagtcattatacgctaattaagtatttgatgaatgactagtgtgacagggctgaggaactccccccgggtaaccggtgcctcagcgtccgaaagacacgtggataggatccaccctgttatacccagcacgatgtaatagtcaaatacctctgatttgtgtaggatgtataaattgtgcattgtaaattttgggcgtagagatgctccgaaggtaccccgttttacgggatctgatcggaggctaattacccaatgcgccctaaataacttcatataatttctttttcttattcaaa 510 Hunnivirustaacgtttggcaagaaccctcacctgtcaattgggaccaccactttcagtgaccccatgcgaagtag A1tgagagagaataagctttcttacccttcatttgtgaacccttcagtcgaagccgcttggaataagataggaggaaaagttcattctaaatggagtgaaacatgtacttcagaatttctagcacgcgctgggctttcttgcgtgtgacggcactgtcttgccggagctctccacactgacaccccacgcttgtggaccttggtggcagatgacaacactgcagctggaattgagtgtctggtacactctgtgtaacagtgaaaacaatgtgatcacttcggtgagctagtagcctgtggaccaacaactggtaacagttgcctcaggggccaaaagccacggtgtttacagcaccctactggtttgattggagcaatccaagatgtcacagagttagtaattgccaagcagtccgtactggtatcttgacataccgtgcagttttggatagtgaaggatgccctgacggtacccataggtaacaagtgacactatggatctaagcaggggctcactctacgctgctttacagctggctgtgagttaaaaaacgtctagctatccacaacctaggggactaggttttccttttatttagattacaattat 511 Hunnivirusacagtttttgacaaggaccctcacctgtcaatcgggaccaccactttcagtgaccccgtgcgaagt A2gttgagagaaagtgagctttcttacccttcatttgtgaacccttcagtcgaagccgcttggaataagatggaaggaaatgttcattctaaatggagtgaaacatatacttaatttccagtgtttagtggtctttccactagacaacggcactgtcttgccggaactctacacaccaacattccacgcttgtgggactcaaatgttggatgacacagttgtagctggaactgagtgtttagtgcactctgtgtaacagtgaaaacaatgtgatcacttcggtgggctagtagcctgtggactaacaactggtaacagttgcctcaggggccaaaagccacggtgttaacagcaccctactagtttgattggagcaatccatgatgttacagagttagtaactgccaaacagattgtactggtatcttggcataccgtgcaacttaggatagtgaaggatgccctggcggtacccataggtaacaagtgacactatggatctaaacaggggctcactctacgttgctttacaactagctgtgagttaaaaaacgtctaactatccacaacctaggggactaggttttcctttttatttttatacacaacta 512Ia Io ggtgtccgggtgtgtgggatagacccagatgtgcagtggatgcgagcatttgagtcagagtaggagcaagcccaggggcaaagggaccacattgtgtatcccgaatgaaggatcgagatttctctcctcattacccggtgtcttgtcactgagggggggcccaacagtcttagtcctatactgcctgatagggtcgcggctggccggactcaagtgctatagtcagttgattttcactc 513 TauractttaaaagtcgtgcgtggcttcaccacgcacgatcagtactatcagttaaccactcttgaatatgctcSyndromeaatgaccctattcaacactggtgtctcttagtacattattttagcacttaacgtgcatgagttttgcccatVirusttctttcaaaaaaatgagtattcgaggagacgtcccgctccccgtcttatttcaaccgtagactcgacatctattggtggacatttaattccagtcgccgtaagttgcttctgccccgcgctatattttcttatacttatggttctataggtctggtttaaaacgtaaatagacggcccacaaactatagaacgcgtacccggaacgccaatcccggataagtccctggatatatagatgcaccgcaatataagcctgcagactgtctcatat act514 ABPVcccgtcaaaataacaacttataacacgatgttacccgaagaaaccattttagtgtaacatttaagattagaagtagttcatctaatagagataggcactattagaaggaggccttttctaaaggagccgttagtcagcccagacaagcgcagtactttagaagagagaagttccccgatagcgaccgaaaagacgcgttttccgtgctaactaatttaaatgtgggaacgaatattattattgaaattatgtgagccacgtagcaatcaagtcatgtttttgtcactacgtttactcatctaatgtagataattttgtttaagtacctatttaggtgtcatcccaccagagaagaaataatacgtaccggaacccagagtacaccccttattttaagccttactgggcttctctgttagttagtaatctggcccacgttttgcgttgagtggggtcccaacagtaggaattcgacggacaagtagcaagcgagtcggtaccaattggtttagcatcgaaattactctgggcaggaagttactaaacgagaactttctgcttaaatcccaacgcacaaacaaatagagtaaataaataattata 515 BRAV-2tttgttttgcggctttgccgttgttcgggttttacctgttttcacacagcaaaacaggccttctagtttcgtgcttaaacgagatcatgctcgaactagaactacatagctggtcactggactcataccacaccttgtggagctttatgggaaaggtggctagtgggctgtggaagtgactctgaccacatgcctctcaagtgtgggaaatcacggatcggtgtagcgacgacaacaggccttgggacaccctctccagtaatggagacccaaggggccaaaagccacgcctcgtgccctgttgttcacaaccccagtgcgacccgtgttagtacctatttgcgagaactgtgtctggacagctaaacacaaccctagtgggagactaaggatgcccaggaggtacccggaggtaacaagtgacactctggatctgacctggggagagagggcttgctttacaggcgcctctctttaaaaagcttctatgtctcatcaggcaccggaggccgggcctttttccttttaaaattacactta 516 BRBV-1ccccccctacttaaagatgtacggttttgctgctttcacagagtaaagcagatagaggttctgaactggcaaactttacctcgaaacacgcccgtttttctgctgtgtctcacagactgtcctgtcacacttgtggcggcttgtgacactgtgaacatagtgagaccgaccaagacaacagatcaagtgtttgaacatcgaacgtctaaactggatccgtaactggacatgttagggcaaggacttcccccctggtaacaggagcctggctggccaaaagccccgctcattgagcctagcatgttgtcgaccctggactgttcagatttgttagtacatggaattcacttgtcacggttcttctgaactcggtctctagtatgacagcctaaggatgccctccaggtaccccggggtaacaagtgacacccgggatctgaggaggggactactttacgtagtttaaaaaacgtctaagctgttatggtgaccagaggctggcacctttcacttttaaaattacactactgactacaattgaagtgataacggttttacaggctttcaaactagttacacaagcactgttttcctgacacacacacttt517 ERAV-1aatattggcgcgcgcatttgcgcgcccccccccatttcagccccctgtcattgactggtcgaaggc U188gttcgcaataagactggtcgtcacttggctgactatcgatcaggattagcgcgccctcgcgcggcgggccgtcaagcccgtgcgctgtatagcgccaggtaaccggacagcggcttgctggattacccggtgccattgctctggatggtgtcaccaagctgacaaatgcggactgaacctcacaaagcgacacacctgtggtagcgctgcccaaaagggagcggaactcccccgccgcgaggcggtcctctctggccaaaagcccagcgttaatagcgccttttgggatgcaggagccccacctgccaggtgtgaagtggagtgagtggatctccaatttggtctgttctgaactacaccatctactgctgtgaagaatgccctggaggcaagctggttacagccctgaccaggggccctgcccgtgactctcgatcggcgcagggtcaaaaattgtctaagcagcagcaggaacgcgggagcgtttcttttccctttgtatcgac 518 GFTVatggggaagggtatgacgtgccccttccttcttcggagaactcgctctagtggtctttccacttctggaaaagagtgagtgcacgtgatcaggaccgtcgaagacgacaaatacctggtgctctatctcatagacgtttcacagctgtagcgacccctcagtagcagcggaagccccctcctggtgacaggagcctctgcggccaaaagccacgtggataagatccactgctgagggcggtgcgaccctagcaccctgtgatgcatactagttgtagcgtgccggactattggtctgtcataagacacctgatagagagaccaagaatgtcctggaggtaccccgcgtgcgggatctgaccaggagaccattgcccaatgctttacaacgggtctatggtttaaaaactgtcgcagtctctccaaaccaagtggtcttggttttcaattactttgaatatttcact 519 SAFVttttcgacgtggttggaattgccatcatttccgacgaaagtgctatcatgcctccccgattatgtgatgtV13Ctttctgccctgctgggcggagcattctcgggttgagaaaccttgaatctttttctttggaaccttggttcccccggtctaagccgcttggaatatgacagggttattttcttgatcttatttctacttttgcgggttctatccgtaaaaagggtacgtgctgccccttccttctctggagaattcacacggcggtctttccgtctctcaacaagtgtgaatgcagcatgccggaaacggtgaagaaaacagttttctgtggaaatttagagtgcacatcgaaacagctgtagcgacctcacagtagcagcggactcccctcttggcgacaagagcctctgcggccaaaagccccgtggataagatccactgctgtgagcggtgcaaccccagcaccctggttcgatgatcattctctatggaaccagaaaatggttttctcaagccctccggtagagaagccaagaatgtcctgaaggtaccccgcgtgcgggatctgatcaggagaccaattggcggtgctttacactgtcactttggtttaaaaattgtcacagcttctccaaaccaagtggtcttggttttccaattttgttgaatggcaat 520SAV ggagatctaagtcaaccgactccgacgaaactaccatcatgcctccccgattatgtgatgctttctgP-113ccctgctgggtggagcatcctcgggttgagaaaaccttcttcctttttccttggaccccggtcccccggtctaagccgcttggaataagacagggttatcttcacctcttccttcttctacttcatagtgttctatactatgaaagggtatgtgtcgccccttccttctttggagaacacgcgcggcggtctttccgtctctcgaaaagcgcgtgtgcgacatgcagagaaccgtgaagaaagcagtttgcggactagctttagtgcccacaagaaaacagctgtagcgaccacacaaaggcagcggaccccccctcctggcaacaggagcctctgcggccaaaagccacgtggataagatccacctttgtgtgcggcacaaccccagtgccctggtttcttggtgacacttcagtgaaaacgcaaatggcgatctgaagcgcctctgtaggaaagccaagaatgtccaggaggtaccccttccctcgggaagggatctgacctggagacacatcacatgtgctttacacctgtgcttgtgtttaaaaattgtcacagctttcccaaaccaagtggtcttggttttcactctttaaactgatttcact 521 VHEVaattccttcttcctttctccttggacctcggtcccccggtctaagccgctcggaatatgacagggttattttcacctcttctctcttctacttcatagtgttctatactatgaaagggtatgtgtcgccccttccttcttggagaacgtgcgtggcggtctttccgtctctcgaaaaacgtgcgtgcgacatgcagagtaacgcaaagaaagcagttcttggtctagctctggtgcccacaagaaaacagctgtagcgaccacacaaaggcagcggaaaccccctcctggtaacaggagcctctgcggccaaaagccacgtggataagatccacctttgtgtgcggtgcaaccccagcaccctggtttcttggtgacaccttagtgaaccctcgaatggcaatctcaagcgcctctgtaggaaagccaagaatgtccaggaggtaccccttcctcatggagggatctgacctggagacacatcacacgtgctatacacttgtgcttgtgtttaaaaattgtcacagctttcccaaaccaagtggtcttggttttcccttaacttcgaaaagtcactatggcctgcaaacatggatacccagacgtgtgccct 522 TRVatgcgacgtggttggagattaaaccgactccgacgaaagtgctatcatgcctccccgattatgtgatNGS910gttttctgccctgctgggcggagcattctcgggttgatacaccttgaatccttcatccttggacctcaggtcccccggtctaagccgcttggaatacgacagggttattttccaatcttctcctttctactttcatgagtcctattcatgaaaagggtctgtgctgccccttccttcttggagaatctgcgcggcggtctttccgtctctcgaaaagcgcagatgcagcatgctggaaccggtgaagaaaacagttctttgtggaaacttagagcagacatcgaaacagctgtagtgacctcacagtagcagcggaaccccctcctggtaacaggagcctctgcggccaaaagccccgtggataagatccactgctgtgagcggtgcaaccccagcaccctggttcgatggttgttctctgtggaaccagagaatggtctttctcaagccctccagtagagaagccaagaatgtcctgaaggtaccccgcatgcgggatctgatcaggagaccaatcgtcagtgctttacactggcgctttggtttaaaaactgtcacagcttctccaaaccaagtggtcttggttttcacttttatcaaactgtttc 523 EMCV2aaatactggtcgaaaccgcttgggataagaccggggtttgttaatgtctcaatgttattctccacccaRD1338attgacgtcttttgtcaattggagggcagtgaaaccttgcccttgcttcttgcagaggattcccagtggtctttccgctctcgacaagggaattcatgatccaccaaaagttgtgaagagagcaggtcccatggaagctttctgacgactgatgatgactgtagcgaccctttgcaggcagcggacccccccacctggtgacaggtgcctctgcggccaaaagccacgtgtttaacagacacctgcaaaggcggcacaaccccagtgcctcatcaaaagtctgatgactgtggaaatagtcaaccggcttttcttaagcaaatttggtgtcggggctgaaggatgcccggaaggtaccacactggttgtgatctgatccggggccacagtacatgtgctttacacatgtagctgcggttaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaccacgattacaat 524 EMCV1gtctgctcgatatcgcaggctgggtccgtgactacccactccccctttcaacgtgaaggctacgataJZ1203 gtgccagggcgggtactgccgtaagtgccaccccaaaacaacaacaaaccccccctaacattactggccgacgccgcttggaataaggccggtgtgcgtttgtctatatgttatttcccaccacattgccgtcttttggcaatgtgtgggcccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaaggaagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcggccctttgcaggcagcggaaccccccacctggcgacaggtgcctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctctcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtgcacatgctttacatgtgtttagtcgaggttaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaacacgatgataat 525 EMCV1atgtggtcgaagccacttggaataagaccggcgtgcgcttgtctatatgttacttccaccacattgccAnrB-3741gtcttttggcaatgtgagggcccggaacctggccctgtcttcttgacgaacattcctaggggactttcccctctcgccaaaggaatgtaaggtctgttgaatgtcgtgaaggaagcagttcctctggaagcttcttgaagacaaacagcgtctgtagcgaccctttgcaggcagcggaaccccccacctggtaacaggtgcctctgcggccgaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgcgttggatagttgtggaaagagtcaaatggctttccccaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccactggttgggatctgatctggggcctcggtgcaggtgctttacacctgttgagtcgaggaaaaaaacgtctaggccccccgaaccacggggacgtggttttcctagaaaaccactatgacaat 526 Cosaviruscgtgctttacacggtttttgaaccccacaccggctgtttggcgcagcaggacagtaggtatattttctt D1tcatttctcttttctagccgcgtaggttctatctacgcgggcggagtgatactcccgctccttcttggacaggcggcctccacgctctttgtggatcttaaggctgccaagtcactggtgtttgaagtgaagaatggagagacactagggcgtttcatgtggctttgccagggattgtagcgatgctgtgtgtgtgtgcggatttcccctcgtggcgacacgagcctcacaggccaaaagccctgtccgaaaggacccacacagtggggttgccccgacccctcccttcaaagctttgtgtaaacaaacttttgtttagactttcttaagcttctctcacatcaggccccaaagatgtcctgaaggtaccctgtgtatctgaggatgagcaccaccaactacccggacttgtgggacgtgtcccacagacgcatgtggtattccagccccctccttttgaggagggggcttttgctcgctcagcacaggatctgatcaggagattcatctctggtgctttacaccagagcatggatttaaaaattgcccaaggcctggcaaacaacctaggggactaggttttctctattttaaaagatgtcaat 527CosaviruscgtgctttacacggtttttgaaccccacaccggctgtttggcgcttgcaggacagcaggtttattttcttB1ttaactctctctttctagccacacacgatctatgtgtgtgggcggagtgatactcccgttccttcttggacaggcggcctccacgccctttgtggatcttaaggctaccaagtcactggtgttggaaagtgaagagaaaggagttccttgggaactacatgtggcattgacagaggttgtagcgatgctgtgtgtgtgtgcggattacccccgtggcgacacggaccccacaggccaaaagccctgtccgaaaggacccacacagtggagcaaccccagctcccctcttcaatgttttgtgttagcaaccttggtattattttctctcaagcttccaatacaccgggccccaaagatgtcctgaaggtaccccgtgtatctgaggatgagcaccatcaactacccggacttgttctttcgagaacagacgcatgtggtaacccagccccgatcctaaggggtcggggcttttgctcactcagcacaggatctgatcaggagacctcccccccctgctttacagggggcgggggtttaaaaattgcccaaggcctggcaaataacctaggggactaggttttcctttttattttaaagttgtcaat 528 CosavirusccgtgctttacacggtttttgaaccccacaccggctgtttggcgcttgcaggacagcaggtttattttcA SH1ttatgctctttatttctagccaacagggttctatcctgttgggcggagtgatactcccgttccttcttggacagattgcctccacgatctttgtggatctcaaggtgatcaagtcactggtaaatagagcgaaggttgaggaaacctgaggaatttccatgtggttttgccaggagttgtagcgatgctgtgtgtgtgtgcggatttcccctcatggcaacatgagcctcacaggccaaaagccctgtccgaaaggacccacacagtggagcaatcccagctccctcctacaaagctttgtgagaatgaactcacgtttattcttctttattctctgtttacatcaggccccaaagatgtcctgaaggtaccttgtgtatctgggcatgagcaccatcaactacccggacttgcatttcggtgcagacacatgtggttacccagcccctctgctttggcagaggggcttttgctcgctcagcacgagatctgatcaggagccctttcccagtgtgctttacacctggcggggggttaaaaattgcccaaggcctggcaaaataacctaggggactaggttttccttttattaacaatgtctgtcatt 529MalagasivirusctttattttcttatgtaactcttctttttaagttttattttgcctacttgtgagcttatgcgggaccactgtBcttagacaaccccacatttgtcatgagtaagtacacgcaaccattacgattactttttaaccgtctgaccttttgataacaactgaagttaggcgtgaaacatgcatttataccaaagtagccccgcatttccccactacggtgggggggctaccctactggctaggaactgtagccattatgtgttgcctggctttcaggatctcacaacacaacagttctctcacaatggaatatgggtgagattgcagtgacatgaacaagtatctagtagtacatagactcaagcctagttgcctgcggaacaacatgtggtaacacatgccccagggtccaaaagacaagggttaacagccccactaggtgtctgtgtgtgaagaatacatagtagtgagaatgatctcacctgttagtacagaatgagtatggcaggtgaaggatgtcctacaggtacccattatatggatctgagtaggagaccactagtggtggctttaccgccaggtgagtggataaaaagcgtctagccaagccaacagcactagggatagtgctttctattttattttattttcagtgtat 530 Mosaviruscccccccctcaaattgcaacgatatagctaatggcgagattgagatgctatatcacctccttctaagtt A2atagacctcatctgattgataaggacgtaatttggtcgaaaccgcttggaataagaccgatgcgcgtSZAL6agtcatgatgatgatgtaagatctaggaacttatccaatctgcttatgtctatgtaagtagaggggcaggcctcattgccctaattctttctaccgagtatctgctagggtttctagcggcagaatacaggattgagggatacaagatactactgatcgattgtcgattgggaaacagtagatacttcaaagctaccagtagcgtggactcacagccagcggactacccctcatggtaacatgagcctctgggcccacaaggcacgtcgcaagacctgtgagacggcaaccccagcctagctttgttgaggaaacaagcgataacatgacatgagagaccggaaggattcttgtattgtgagccgaaggatggcctctaggtacctcattttatgagatctgaggaggtgctcttgagttggtgctttacactgcacaacacagagttaaaaagcgtctaagctcacccggaaattgggaaatttccgttatttccattttgtttgcaaagtcgttc 531 SVVctgggccctcatgcccagtccttcctttccccttccggggggtaaaccggctgtgtttgctagaggcacagaggagcaacatccaacctgctttttgtggggaacggtgcggctccaattcctgcgtcgccaaaggtgttagcgcacccaaacggcgcatctaccaatgctattggtgtggtctgcgagttctagcctactcgtttctcccctatccactcactcacgcacaaaaagtgtgctgtaattacaagatttagccctcgcacgagatgtgcgataaccgcaagattgactcaagcgcggaaagcgctgtaaccacatgctgttagtcccttcatggctgcgagatggctatccacctcggatcactgaactggagctcgaccctccttagtaagggaaccgagaggccttcttgcaacaagctccgacacagagtccacgtgattgctaccaccatgagtacatggttctcccctctcgacccaggacttctttttgaatatccacggctcgatccagagggtggggcatgatccccctagcatagcgagctacagcgggaactgtagctaggccttagcgtgcttaggatactgcctgatagggcgacggcctagtcgtgtcggttctataggtagcacatacaaat 532 PTV Aactagtccttggacttttgttgtgtttaaacacagaaatttaattacctggccatgaattcattggattaaccccttctgaaagacttgctctggcgcgagctaaagcgcaattgtcaccaggtattgcaccagtggtggcgacagggtacagaagagcaagtactcctgaccgggcaatgggactgcattgcatatccctaggcacctattgagatttctctggggcccaccggcgtggagttcctgtatgggaatgcaggactggacttgtgctgcctgacagggtcgcggctggccgtctgtactttgtatagtcagttgaaactcacc 533PTV Bcttccttttaattcgtaactgataagtgatagtccttggaagctaggttttagttacgctagttttggattatcttgtgcccaacatttgttttcgaacatatgttgtgtttaaacacagaaatctagtttctttggttatgagtttaatggaatatccttttgaaagacttgccttggcgcgggctagagcgcaattgtcaccaggtattgcaccaatggtggcgacagggtacagaagagcaagtactcctgactgggtaatgggactgcattgcatatccctaggcatctattgagatttctctggagcccaccagcatggagacctgtatgggaatgcaggactggacttgtgctgcctgacagggtcgcggctggccgtctgtactttgtatagtcagttgaaactca tt534 Tottoriviruscccctttacgtaactgcaacttaaagagtaccctactgcattggatgtgtggtaaacttttacgcacacatttgtagtagtgttagttatgttctacctaatgagtatgcatgcacccgtcgaaacacgcttgtgataagataggtgagtccatgtgactaatctcattaagataaataagcaccctacaacgcacggcacgctcgtgtcttccgtgcggggccgggacaacagcggcctaaatcttctaggtgaccaccatgcttttgggactatggcaccactgtggacgtgagtacctggcagtaagtctgtgaaaagatggaaggtgtcccaagctatggggcgtatgcatatagcctgcggaacaaacaacggcgacgttgtccccagggcccaaaaggcacgtggataagatccacctatatgtttaccccatagtgtaagtcactggaagtcctagtaatggatgtctggagtaaggctcacggggtagggcgaaggatgcccagaaggtacccgtaggtaaccttaagagactatggatctgatctggggaccggatggcgccatcaccatgacgtggaggccggtttaaaaaacgtctaagcccgaccaacaacctaggggactaggttttccttttttattcatgtatgacgtt 535Posavirusacatttccttgcgtgcgcacccgaaaatttattgaacttggcttgaatcataagtaatgcttcttatagc 1ggacactttgagaatataatgatgtatggattaatgtatactgttttaaataacaaacctagacacgcagttgcgttgatggttgtatcaatcacatataagtgttgaactcgtgttaatcctcgatcgctatatgtttgccgcctacttccaataaaatagattacatgcgcgtcatgcctttgtgggttacctattggcctctgacaaaaacaagtcgtaagatttgtagcttcccggtgtaaaaagctgggcgcggtctggctctcgtagggtggaaaggtccaccaatggctggttgagtgtaagctccggtgtcctggttgtcgcaattccaggcgtcgtaataacctatattgcatctgactctaactcttgtggctctactgtatctagttcttgttctactaactctaataatactactggctctaatactgaaaaacttacatatgttaatatagataatatccttgatcctgatatccctcacgtcactgaagttcgccgaaaacgaatttcagatcatatcattgaatctcaaggatgtacttgctctgaacctactataactcctcatgcgttttcattttctactcttggc 536 A105-675ccacccacagcaagaatgccatcatctgtcctcacccccatttctcccctccacccctgcaaccattacgcttactcgcatgtgcattgagtggtgcacgtgttgaacaaacagctacactcacgtgggggcgggttttcccgccatcggcctctcgcgaggcccaccatccctttcctcccataactacagtgctttggtaggtaagcatcctgatcccccgcggaagctgctcgcgtggcaactgtggggacccagacaggttatcaaaggcacccggtctttccgcctccaggagtatccctgctagtgaattctagtggggctctgcttggtgccaacctcccccaaatgcgcgctgcgggagtgctcttccccaactcaccctagtatcctctcatgtgtgtgcttggtcagcatatctgagacgatgttccgctgtcccagaccagtccagcaatggacgggccagtgtgcgtagtcgtcttccggcttgtccggcgcatgtttggtgaaccggtggggtaaggttggtgtgcccaacgcccgtactttggtgacaactcaagaccacccaggaatgccagggaggtaccccgcctcacggcgggatctgaccctgggctaattgtctacggtggacttcttgcaccatttctttcttctgttc 537 A110-675acctttgtgcgcctgttttataccccctcccccaactgtaacttagaagtaacacacaccgatcaacagtcagcgtggcacaccagccacgttttgatcaagcacttctgttaccccggactgagtatcaatagactgctcacgcggttgaaggagaaagcgttcgttatccggccaactacttcgaaaaacctagtaacaccgtggaagttgcagagtgtttcgctcagcactaccccagtgtagatcaggtcgatgagtcaccgcattccccacgggcgaccgtggcggtggctgcgttggcggcctgcccatggggaaacccatgggacgctctaatacagacatggtgcgaagagtctattgagctagttggtagtcctccggcccctgaatgcggctaatcctaactgcggagcacacaccctcaagccagagggcagtgtgtcgtaacgggcaactctgcagcggaaccgactactttgggtgtccgtgtttcattttattcctatactggctgcttatggtgacaattgagagatcgttaccatatagctattggattggccatccggtgactaatagagctattatatatccctttgttgggtttataccacttagcttgaaagaggttaaaacattacaattcattgttaagttgaatacagcaaa 538 18-675cccacagcaagaatgccatcatctgtcctcacccccaattttcccttttcttcccctgcaaccattacgcttactcgcatgtgcattgagtggtgcatgtgttgaacaaacagctacactcacatgggggcgggttttcccgccctacggcctctcgcgaggcccaccccttccctccccttataactacagtgctttggtaggtaagcatcctgatcccccgcggaagctgctcacgtggcaactgtggggacccagacaggttatcaaaggcacccggtctttccgccttcaggagtatccctactagtgaattctagcggggctctgcttggtgccaacctcccccaaatgcgcgctgcgggagtgctcttccccaactcaccctagtatcctctcatgtgtgtgatggtcagcatatctgagacgatgttccgctgtcccagaccagtccagtaatggacgggccagtgcgtgtagtcgtcttccggcttgtccggggcatgtttggtgaaccggtggggtaaggttggtgtgcccaacgcccgtactttggtgacacctcaagaccacccaggaatgccagggaggtaccccacctcacggtgggatctgaccctgggctaattgtctacggtggttcttcttgcttccacttctttcttctgttcacg 539 A115-675acctttgtgcgcctgttttataccccccccaacctcgaaacttagaagtaaagcaaacccgatcaatagcaggtgcggcgcaccagtcgcatcttgatcaagcacttctgtaaccccggaccgagtatcaatagactgctcacgcggttgaaggagaaaacgttcgttacccggctaactacttcgagaaacccagtagcatcatgaaagttgcagagtgtttcgctcagcactacccccgtgtagatcaggccgatgagtcaccgcacttccccacgggcgaccgtggcggtggctgcgttggcggcctgcctatggggcaacccataggacgctctaatacggacatggtgcgaagagtctattgagctagttagtagtcctccggcccctgaatgcggctaatcctaactgcggagcacatacccttaatccaaagggcagtgtgtcgtaacgggtaactctgcagcggaaccgactactttgggtgtccgtgtttccttttaatttttactggctgcttatggtgacaattgaggaattgttgccatatagctattggattggccatccggtgactaacagagctattgtgttccaatttgaggatttaccccgctcacactcacagtcgtaagaacccttcattacgtgttatttctcaactcaagaaa 540 A73-675ttactccattcagcttcttcggaacctgttcggaggaattaaacgggcacccatactccccccaccccccttttgtaactaagtatgtgtgctcgtgatcttgactcccacggaacggaccgatccgttggtgaacaaacagctaggtccacatcctcccttcccctgggagggcccccgccctcccacatcctccccccagcctgacgtatcacaggctgtgtgaagcccccgcgaaagctgctcacgtggcaattgtgggtccccccctcatcaagacaccaggtctttcctccttaaggctagccccggcgtgtgaattcacgttgggcaactagtggtgtcactgtgcgctcccaatctcggccgcggagtgctgttccccaagccaaacccctggcccttcactatgtgcctggcaagcatatctgagaaggtgttccgctgtggctgccaacctggtgacaggtgccccagtgtgcgtaaccttcttccgtctccggacggtagtgattggttaagatttggtgtaaggttcatgtgccaacgccctgtgcgggatgaaacctctactgccctaggaatgccaggcaggtaccccacctccgggtgggatctgagcctgggctaattgtctacgggtagtttcatttccaatccttttatgtcggagtc 541 Kobuvirusttactccattcagcttcttcggaacctgttcggaggaattaaacgggcacccatacacccccatccc16317ctttctgcaacttaagtatgtgtgctcgtgatcttgactcccacggaatggatcgatccgctggagaacaaactgctagatccacatcctcccttcccctgggaggaccttggtcctcccacatcctccccccagcctgacgtaccacaggctgtgtgaagcccccgcgaaagctgctcacgtggcaattgtgggtccccccttcatcaagacaccaggtctttcctccttaaggctagccccgatgtgtgaattcacattgggcaactagtggtgtcactgtgcgctcccaatctcggccgcggagtgctgttccccaagccaaacccctggcccttcactatgtgcctggcaagcatacctgagaaggtgaccgctgtggctgccagcctggtaacaggtgccccagtgtgcgtaaccttcttccgtcttcggacggtagtgattggttaagatttggtgtaaggtccatgtgccaacgccctgtgcgggatgaaacctctactgccctaggaatgccaggcaggtaccccacccccgggtgggatctgagcctgggctaattgtctacgggtagtttcatttccaattcttttatgtcggagtc 542 AichivirusttactccattcagcttcttcggaacctgttcggaggaattaaacgggcacccatacatccccatccccChshc7tttctgtaacttaagtatgtgtgcttgtaatcttgactcccacggaatggatcgatccgctggagaacaaactgctagatccacatcctcccttcccctgggaggaccttggtcctcccacatcctccccccagcctgacgtaccacaggctgtgtgaagcccccgcgaaagctgctcacgtggcaattgtgggtccccccttcatcaagacaccaggtctttcctccttaaggctagccccgatgtgtgaattcacattgggcaactagtggtgtcactgtgcgctcccaatctcggccgcggagtgctgttccccaagccaaacccctggcccttcactatgtgcctggcaagcatatctgagaaggtgttccgctgtggctgccagcctggtaacaggtgccccagtgtgcgtaaccttcttccgtctccggacggtagtgattggttaagatttggtgtaaggttcatgtgccaacgccctgtgcgggatgaaatctctactgccctaggaatgccaggcaggtaccccaccctcgggtgggatctgagcctgggctaattgtctacgggtagtttcatttccaatccttttatgtcggagtc 543 AichivirusactccattcagcttcttcggaacctgttcggaggaattaaacgggcacccatacacccccatcccctGoianiatttttgcaacttaagtatgtgtgctcgtaatcttgactcccacggaatggatcgatccgctggagaacaaactgctagatccacatcctccctcccccctgggaggacctcggtcctcccacatcctccccccagcctgacgtatcacaggctgtgtgaagcccccgcgaaagctgctcacgtggcaattgtgggtccccccttcatcaagacaccaggtctttcctccttaaggctagtcccgatgtgtgaattcacatcgggcaactagtggtgtcactgtgcgctcccaatctcggccgcggagtgctgttccccaagccaaacccctggcccttcactatgtgcctggcaagcatatctgagaaggcgttccgctgtggctgccagcctggtaacaggtgccccagtgtgcgtaaccttcttccgtccccggacggtagtgattggttaagacttggcgtaaggttcatgtgccaacgccctgtgcgggatgaaacctctactgccctaggaatgccaggcaggtaccccaccttcgggtgggatctgagcctgggctaattgtctacgggtagtttcatttctaattctttcatgtcggagtc 544 AichivirusttactccattcagcttcttcggaacctgttcggaggaattaaacgggcacccatacacccccaccccETHP4ctttttgcaacttaagtatgtgtgctcgtgatcttgactcccacggaatggatcgatccgctggagaacaaactgctagatccacatcctcccacccttgggaggacctcggtcctcccacatcctccccccagcctgacgtaccacaggctgtgtgaagcccccgcgaaagccgctcacgtggcaattgtgggtccccccttcattaagacaccaggtctttcctccttaaggctagtcccgatgtgtgaattcacattgggcaactagtggtgtcactgtgcgctcccaatctcggccgcggagtgctgttccccaagccaaacccctggcccttcactatgtgcctggcaagcatatctgagaaggtgttccgctgtggctgccagcctggtaacaggtgccccagtgtgcgtaaccttcttccgtcttcggacggtagtgattggttaagatttggcgtaaggttcatgtgccaacgccctgtgcgggatgaaacctctactaccctaggaatgccaggcaggtaccccaccctcgggtgggatctgagcctgggctaattgtctacgggtagtttcatttccaattcttctatgtcggagtc 545 AichivirustactccattcagcttcttcggaacctgttcggaggaattaaacgggcacccatacacccccacccccDVI2169ttttctgcaacttaagtatgtgtgctcgtaatcttgactcccacggaatggatcgatccgctggagaacaaactgctagatccacatcctcccttcccctgggaggaccccggtcctcccacatcctccccccagcctgacgtatcacaggctgtgtgaagtccccgcgaaagctgctcacgtggcaattgtgggtccccccttcatcaagacaccaggtctttcctccttaaggctagccccgatgtgtgaattcacattgggcaactagtggtgtcactgtgcgctcccaatctcggccgcggagtgctgttccccaagccaaacccctggcccttcactatgtgcctggcaagcatatctgagaaggtgttccgctgtggctgccagcctggtaacaggtgccccagtgtgcgtaaccttcttccgtctccggacggtagtgattggttaagatttggtgtaaggttcatgtgccaacgccctgtgcgggatgaaacctctactgccctaggaatgccaggcaggtaccccaccttcgggtgggatctgagcctgggctaattgtctacgggtagtttcatttccaattcattttatgtcggagtc 546 AichivirusgcttcttcggaacctgttcggaggaattaaacgggcacccatacacccccacccccttttctgcaacDVI2321ttaagtatgtgtgctcgtaatcttgactcccacggaatggatcgatccgctggagaacaaactgctagatccacatcctcccttcccctgggaggaccccggtcctcccacatcctccccccagcctgacgtatcacaggctgtgtgaagtccccgcgaaagctgctcacgtggcaattgtgggtccccccttcatcaagacaccaggtctttcctccttaaggctagccccgatgtgtgaattcacattgggcaactagtggtgtcactgtgcgctcccaatctcggccgcggagtgctgttccccaagccaaacccctggcccttcactatgtgcctggcaagcatatctgagaaggtgttccgctgtggctgccagcctggtaacaggtgccccagtgtgcgtaaccttcttccgtctccggacggtagtgattggttaagatttggtgtaaggttcatgtgccaacgccctgtgcgggatgaaacctctactgccctaggaatgccaggcaggtaccccaccacgggtgggatctgagcctgggctaattgtctacgggtagtttcatttccaattcttttatgtcggagtc 547Aichivirustactccattcagcttcttcggaacctgttcggaggaattaaacgggcacccactttcctgtcctctcccrat08cttttctgtaactccaagtgtgtgctcgtaatcttgactcccgcggattgaccgctccgctggtgaacaaactgctaggtcatctcctccccacccttgggcgtccttccgggcgtccacaccctccccccagcctgacgtgtcacaggctgtacaaagaccccgcgaaagctgctaacgtggcaattgtgggtcccccctttgtaaaggaaccgagtctttctcccttaaggctagacccctgtgtgaattcacaggtggcaactagtggaccactgcatgctcccgacctcggccgcggagtgctgttccccaagtcgtaacactgaccacacttatgtgcctggcaagcatatctgagaagatgttccgctgtggctgccaaacctggtaacaggtgccccagtgtgcgtagtcttcttccgtcttcggacggtaggtgttaggtaaagatgcggcgtaaggttcaagtgccaacgccctggaagggatgacccttctactgccctaggaatgccgcgcaggtaccccaggttcgcctgggatctgagcgcgggctaattgtctacgggtagtttcatttccctcttcttccactggcatc 548 AichivirusactccattcagcttcttcggaacctgttcggaggaattaaacgggcacccactttcctgtcctctcccRt386 cctttctgcaactcaagtgtgtgctcgtaatcctgactcccacgggagaccgccccgttggtgaacaaacagctaggtcattccctccctacccctgggcgccatttcagtggcgttcatatcctccccccagcctgacgtgtcacaggctgtgcaaagtccccgcgaaagctgctcacgtggcaattgtgggtcccccctttgtgaaggaaccgagtctttctcccttaaggctagacccctgtgtgaactcacaggtggcaactagtggttccactgcatgctcccgacctcggccgcggagtgctgttccccaagtcgtgacactgacctccacttatgtgcctggcaagcatatctgagaagatgttccgctgtggctgccaaacctggtaacaggtgccccagtgcgtgtagtcttcttccgtctccggacggtaagtgtgtggtaaagatgcggcgtaaggttcaagtgccaacgccctggaagggatgacccttctactgccctaggaatgccgcgcaggtaccccaggttcgcctgggatctgagcgcgggctaattgtctacgggtagtttcatttccctctcttttcactggcatc 549 Norwaygtataagggttgggaaccttgtaccaagctacctctgccattcagtatttgggagtagaagtagatgt RatgtttacaaactcacacgtgtgggggcggggatagactgtgccagcggtcgtgtaccagcacctacPestivirusgcatacgtgtggactgcgaaccaggagagcacctaggtctgacaagctgtgagaacacagtagtcgtcagtgagtcagctggtaaggatcacccacctggatactcacgtggacgagggagtttcccagtcagaaacctacaccagaggaggggtcctctggagacatggatggtctgagtaacagactatctactggggtgtgctgcctgacagggtctcggctgatagcctggctagcagtataaaaatcagttgaattggcatatgagttgtgaacatctagtaaacaatgaaagacaaaaacaaaaaatgagcataataaaaaaattgtacaatccactactcaggtgtggctgcagactt 550 PorcinetttgaaaagggggtgggggggcctcggccccctcaccctcttttccggtggccattcgcccgggcKobuviruscaccgttactccactccactccttcgggactggtttggaggaacacaacagggcttcccatccctgt GS2ttaccctttattccatcatcctttccccaagtttaccctatccacaccccactgactgactcctttggattttgacctcagaatgcctatttgacctcccactcgcctctcccttttcggattgccggtggtgcctggcggaaaaagcacaagtgtgttgcaggctaccaaactcctacccgacaaaggtgcgtgtccgcgtgctgagtaatgggataggagatgcctacaacaggctcgcccatgagtagagcatggactgcggtgcatgtgacttcggtcaccacgggcatagcattgctcacccgtgaatcaagtcatcgagatttctctgacctctgaagtgcactgtggagcgtggctgggaatccacgcttgaccatgtactgcttgatagagtcgcggctggccgactcatgggttaaagtcagttgacaagacac 551 KobuviruscctacccaagggttacatgggaccatattcctcctcccctgtaactttaagttttgtgcccgtattcagSZAL6actccaggcggatgttgtgtcgcccgtcctgtgaacaaacagctagacactttcctcccctccctctgggctgctccggcagtccactccctccccccagcgtaacatgccccgctggagtgatgcacctggaagtcgtggacgtgggttagtaacttcggtgaaaacccactataatgacaactggttgacccccacactcaaaggactcgagtctttctcccttaaggctagcccggccacatgaatttgcagctggcaactagtgagtccaccatgtcccgcaacctcggctgcggagtgctgttccccaagcgtatgccttccttctgtaagagtgcgcctggcaagcacatctgagaagtcgttccgctgcgtcgtgccaacctggcgacaggtgacccagtgtgcgtagacttcttccggattcgtccggctcttctctaggaaacatgcgtgtaaggttcatgtgccaaagccctgcgcgcggtgttcttctactgccctaggaatgtgccgcaggtacccctacttcggtagggatctgagcggtagctaattgtctacgggtagtttcatttccatcttctcttcaggtcgacatc 552 Kobuvirus gaccttctggtacttcttcgcctgggtcacaaaagcgaagaacctgcctctctaacgccagacgagsheep TB3cggcattaaacttgaacttctggcactctccactctcccttttccctgtccctttccccactgcgctctcaaggtcgcgcaatcctgggactagcccagttttaaaagttcctggcaccctttgcccctctaggcccttaaggtaggaactgaccttgtgctgtgatctcggtgcgggagtgctaccacgtagtcatcgtaagcctcgtttctggactgccctggcaaggctacagagtaccgtgttccgctgtggatgccatccgggtaaccggacccccagtgtgtgtagcggtatgttcacggtccgccgtgttcaccagattcctgacctggctttgctagaaatggtgtgtgcccaatccctgtgaccagtatcaattacatcacctaggaatgctaggaaggtaccccagtcctgagctgggatctgatcctaggctaattgtctacggtgatgctccttttattttcttacaactgctattgactgtctgattgctgattctgctcttgtgctcttctgctctggctcattctcaagggttctctttgtccaagatcctttggttctctccttgttccacttgccactgccaacgcttgtc 553Pronghorngtatacgcagttagttcatcctgtgtatacagattggagactctaaaaacaacgattcggaatagggantelope gcccgcggcgaagaccgaagacaggctaaccatgccgttagtagggctagcaccaaaacgcgpestivirusggaactagacacttaggagagtggtctggctactctaagaggtgagtacaccttaaccgtcaagggttctactcctcagagaggactagagatgccctgtggacgggggcatgcccaagagttagcttagccggggcgggggttgttccggtgaaagtagcaatattgaccacactgcctgatagggcggagcaggccccctaggtagtctagtataaaatgtctgctgtacatggcac 554 Porcinetacgcggggtataacgacagtagacaagtgtcgttatgcatcattggccataacaaattatctaatttpestivirusggaatagggacctgcgacctgtacgaaggccgagcgtcggtagccattccgactagtaggacta isolategtacaaataggtcaactggttgagcaggtgagtgtgctgcagcggctaagcggtgagtacaccgtBungowannahattcgtcaacaggtgctactggaaaggatcacccactagcgatgcctgtgtggacgaggacatgtccaagccaatgttatcagtagcgggggtcgttactgagaaagctgcccagaatgggtagttgcacatacagtctgataggatgccggcggatgccctgtattttgaccagtataaatattatccgttgtaaagcat555 Porcinegcagatatcggtggtggacctgggggttgggctcaccgtgccccttcatggggtagacctcactgpestivirus cttgatagagtgccggcggatgcctcaggtaagagtataaaatccgttgttcactaac 1556 Pestivirusgtatacgagtttagctcaatcctcgtatacaatattgggcgtcaccaaatatagatttggcataggcagiraffe-1acaccccgatgcgaaggccgaaaagggctaaccatgcccttagtaggactagcaaaaaatcggggactagcccaggtggtgagcttcctggatgaccgaagccctgagtacagggcagtcgtcaacagttcaacacgcagaataggtttgcgtcttgatatgctgtgtggacgagggcatgcccacggtacatcttaacctatccgggggtcggataggcgaaagtccagtattggactgggagtacagcctgatagggtgttgcagagacccatctgataggctagtataaaaaactctgctgtacatggcac 557 Classicalgtatacgaggttagttcattctcgtatgcatgattggacaaattaaaatacaatttggatcagggcctcswine cctccagcgacggccgaactgggctagccatgcccacagtaggactagcaaacggagggactafever virusgccgtagtggcgagctccctgggtggtctaagtcctgagtacaggacagtcgtcagtagttcgacgtgagcagaagcccacctcgatatgctatgtggacgagggcatgcccaagacacaccttaaccctagcgggggtcgctagggtgaaatcacaccacgtgatgggagtacgacctgatagggtgctgcagaggcccactattaggctagtataaaaatctctgctgtacatggcac 558 Humantcagggttggtaggtcgtaaatcccggtcaccttggtagccactataggtgggtcttaagagaaggtpegivirustaagattcctcttgtgcctgcggcgagaccgcgcacggtccacaggtgttggccctaccggtgggisolate aataagggcccgacgtcaggctcgtcgttaaaccgagcccgtcacccacctgggcaaacgacgcJD2B1I ccacgtacggtccacgtcgcccttca 559 Humancccggcactgggtgcaagccccagaaaccgacgcctatttaaacagacgttatgaaccggcgccpegivirusgacccggcgaccggccaaaaggtggtggatgggtgatgccagggttggtaggtcgtaaatcccg isolategtcatcttggtagccactataggtgggtttaaagggaggtcaaggtccctctggcgcttgtggcgaGBV-C-ZJgaaagcgcacggtccacaggtgttggccctaccggtgtgaataagggcccgacgtcaggctcgtcgttaaaccgagcccattacccacctgggcaaacaacgcccacgtacggtccacgtcgccctacaatgtctctcagaccaataggctttgccggcgagagacaaggaccagtgggggctgggcgacgggggtcgtataggaagaaaaatgccacccgccctcacccgaaggttcttgggctaccccggctgcaggccgccgcggagctggggtagcccaagaaccttcgggtgagggcgggtggcatttttcttcctataccgatc 560 Humantggtcaccttggtagccactataggtgggtcttaagagaaggaaagattcctcttgtgcctgcggcgpegivirusagaccgcgcacggtccacaggtgttggccctaccggtgtgaataagggcccgacgtcaggctcg isolatetcgttaaaccgagcccatttcccgcctgggcaaacgacgcccacgtacggtccacgtcgcccttttJD2B8C aatgtctctcttgaccaataggttcatccggcgagagacaaggaccagtgggggccgggggtcacagggatggaccctgggccctgcccttcccggcggggtggggaaagcatggggccacccagctccgcggcggcctgcagccggggtagcccaagaaccttcgggtgagggcgggtggcatttttcttcctataccgatc 561 Hepatitisgccgggtggaaggcccggaaccgccccaccacctcaactaggtggtaagggtacgtctatcggt GBccggctggcccgaaaggcggtggatcctgtgtgttagggttcgtaggtggtaaatcccagcacagvirus Agtggtaatcgctatagggcaggcttatcccggtgaccgcttccctggatcctggagcgggtcgtggcggcacggtccacaggagtggggcctccggtgtgaataagccctcgtctggagcatcagacgttaaactgagacgtcccgaagagatcggaacgacgccccacgtatggcaacgccgcttaaaacccttcggggacagctatgcgggttgacaatgccagtggggggccgggcccactattgagtgggctccgagttcctctagggatggccgaaaggcagccatggggccacccaggcggcgccgtgctacaggcggcaaggggaaaaatccttcgggtgaccccgggtggcattccctcccttagcagcatgagtgtggtggtagctgcaacc 562 Simianggggaatctcaccccccgtccggttccggaagaatcggaaaccgacaccctgaccaatcattcttpegivirusgatcatagagtggatgaagtgaaagccagacgaaagccggcggatgggtggtgacagggttggtaggtcgtaaatcccggccaccctggtacccggtataagttgggcggaagctgactgaagctccgtgctcttttctgtgcgttcttggtgcacggtccacaggtgacgcctataccggtgtgaataataggccgactcgagcggagtcgttaaactgagaacctccatacggatggcaacttggcttgcgtacggggacgccgctaaagtcacagtgggttaagtccggcgggttgacaaccccagcaaggcgagggggtcctattgttggactctgccagacccggtggaggtaggcatggggtggcccagctccgcggcgcgctacagccggggtagcccaaaatccgaaaggtgagggcgggccacatgtccgaaatttagtcaag c 563Pegivirusagaatggtctaagtggttgccaccgtggtccgaaggggaggaggacctacgctgccagggttgg Icaggtcgtaaatcccgggtgtaggagatccctccttgttaggactgctggtagctggggggtcggtgaccccctgggcaaccgccaaacccggacgaccgggtggcggctccatgttggcacggtccacaggtgtgaaccctaccggtgtgaataagggttggtggttgcggtccaccttaaacgtagtatgcattgggcttggtaaaacaccgctcgtagtacggaacgccgcctttaaagacacagtaggcgtagccggcgggttgacaatccatacggggggtggggtgtggtcatggatctgtccacaccaccttcatgcggccctctaagcaagccataccggggggaggcgcgcggcaccgcactgccgggcaaggggaagaaccttcgggtgacccccccccaaccaccgtccgatcaatgctaatgttgcgtttaggcgtgacaccggcaca 564 Pegivirusagaatggtgtgatccgtcgccgctccagcggaaagcgggcgggatctagtggttagggttgttcg KcgtaaatcccacactagtggtacgctcgtataacgtgggagcagccggTggggtcgaccccccAcctggcggctgctgagcaccggacgaagcgcggggggtgaacgctaacccgcggcccgggctgccaacgttaggcacgtcaggctggaagacgttaaacacagggccccccctcaaccctgatccgaggccagagaccaaggtacgccgcccctttaaaggcgttactcgtccaataggatctctccggcgggttgtcaaaccttgctggccctggtgatggttacgggagggggtggggcggggagtagaagccccgcccggcatgggggtaccaagctcggcacgcccagcacgcgtggcgtaggggaaaaatccttcgggtgacccctggtaccataaagtaattaacatgagcatgccgctagggtgtgctttttcttccttccttgggaaggcggtggcacc 565 Theiler'stgataccgtgtcccggtacgacctcgcgcgtccccaagctcgccctgaggggggagcgtaagggdisease-cgcgtagtggggtagccccccaaaccgagccaccctagtgagtgactttagaatggttagggagaassociatedctaccgccttcgctgtttggggacctaatgatccgcgtgccagggttcttcgggtaaatcccggcgcvirus ggtgttttgggttcagggcagtaggggcagacgggccagcagtcgctggttcctggtaccaccaccctatccggacgacctccctcacgaaaggtcgccacggtctgtggctcgacgacgcctataattcagtccgaggggcgcagccctcgttaaacttaggcaaggttcctcgccattgatttggccaggggtttaagtgaacgccgcccttttaatgtttaatagggttctttcccggcgggttgacaaacacttccctgggctcttcgttggcctcggttccttgatgcttcggcacccatgagcgcacaggggggggaccctgcgacagtccgccaagaggaaaatccttcgggtgacctcgtgcgcaacccaatcccttcttcttccacatggcgtgtctgtggtgcatgctgtg 566 Rodentggacttcggtccccctgttactctgcgagccaccgcagagccagggttggtacgcccgaggtgttpegivirusagaccccggccgaaagctcctaaccatggggttagtaggacgtggtaaatgccactgaggggttggagagctggtagagcgagtaagtcggcgtaaggcccgagtacgggcctcccagcccgggtcagcctaaacctggctgtgatacccggtgcatggagggcgtgtcccaacgctcgatcgctgtagggtgggtccctgcagttgggtgtggctaccctgctcgtactgcttgatagagtcccggcggacggaccagctctcgtcagtccgtggagttgcac 567 Humanaactgttgttgtagcaatgcgcatattgctacttcggtacgcctaattggtaggcgcccggccgaccpegivirusggccccgcaagggcctagtaggacgtgtgacaatgccatgagggatcatgacactggggtgag 2cggaggcagcaccgaagtcgggtgaactcgactcccagtgcgaccacctggcttggtcgttcatggagggcatgcccacgggaacgctgatcgtgcaaagggatgggtccctgcactggtgccatgcgcggcaccactccgtacagcctgatagggtggcggcgggcccccccagtgtgacgtccgtggag cgcaac568 GB viruscccccggcactgggtgcaagccccagaaaccgacgcctatctaagtagacgcaatgactcggcgC/Hepatitisccgactcggcgaccggccaaaaggtggtggatgggtgatgacagggttggtaggtcgtaaatcc G viruscggtcaccttggtagccactataggtgggtcttaagagaaggttaagattcctcttgtgcctgcggcgagaccgcgcacggtccacaggtgttggccctaccggtgggaataagggcccgacgtcaggctcgtcgttaaaccgagcccgttacccacctgggcaaacgacgcccacgtacggtccacgtcgcccttcaatgtctctcttgaccaataggcgtagccggcgagttgacaaggaccagtgggggccgggggcttggagagggactccaagtcccgcccttcccggtgggccgggaaatgc 569 EquineagaatggggagttaactcctggcactggcccgaagcatgaactgatcgcggtggcagggttcttcPegivirusgggtaaatcccggccgcgtgttgtgattgtgttagggcaggtgacagtcggcagggtcgaccccc 1tgcttcaggaccactgtcttcctggacgaccgttgctgaaaaagggccgccacggtctgtagctcgccgacgcttctaattcaggccggaggaccacgctccgtaatcgagcccaagtactcaaaccccaggacaaagggcgcagggttacctggtactacgagcttgggtgtccctgggagtaatcccagggtgc c 570Culexatataaatcccagtttggttaaacctatttcaaggcttaagttgtttattattttatcgccgctcgtgatheilerictataaagttgcctagcggagagagataaagaagaaggagttcaaggctcagggcagggcgcaagttflavivirusccctggtccctaggccgctcgcaggaaggaggagtgaagaagaagaaagagaaggagaggaccaccgccgaaagaaggcaggtgcctcacaagagggccaaccagcgtgttggaccagtggccaacgccggacggcgtggtggcctgctgggacgcctggggattggatggagtgccttcctacaggaagacatcgttcaagccatc 571 Bussuquaraagtatttcttctgcgtgagaccattgcgacagttcgtaccggtgagttttgacttaacgcagtgagaavirus aagttttcgaggaaagacgagaagcgaattctctga 572 Zika Virusagttgttgatctgtgtgagtcagactgcgacagttcgagtctgaagcgagagctaacaacagtatcaacaggtttatttggatttggaaacgagagtttctggtc 573 Yokose virusagtaaattttgcgtgctagtcgctgagcgtcagaccgcaaagtgagtttttagtgatctaaagtgaggagttattcttactgtcatcaaacactacaaataaacacgttgaaattatttccggaagaacaactgtccggaataaagacg 574 Wesselsbronagtatattctgcgtgctaatcgttcgacgttagtccgtggagtgagcttctattagagtcgttaacacgvirus tttgaataatttctactgaaaggagtagaagaaaggagattcattccca 575 Equineacctccgtgctatgcacggtgcgagtcagcgttttgcgcagctttgcgctacacgcgtcgtccaachepacivirusgcggagggattcttccacattaccatgtgtcactccccctatggagggaccaccccgcccacacggaaataggttaaccatacctatagtacgggtgagcgggtcctcctagggcccccccggcaggtcgagggagctgaaattcgtgaatccgtgagtacacggaaatcgcggcttgaacgtcatacgtgaccttcggagccgaaatttgggcgtgccccacgaaggaaggcgggggcggtgttgggccgccgccccctttatcccacggtctgataggatgcttgcgagggcacctgccggtctcgtagaccataggac 576Hepacivirusaccacaaacactccagtttgttacactccgctaggaatgctcctggagcaccccccctagcagggc Bgtgggggatttcccctgcccgtctgcagaagggtggagccaaccaccttagtatgtaggcggcgggactcatgacgctcgcgtgatgacaagcgccaagcttgacttggatggccctgatgggcgttcatgggttcggtggtggtggcgctttaggcagcctccacgcccaccacctcccagatagagcggcggcactgtagggaagaccggggaccggtcactaccaaggacgcagacctctttttgagtatcacgcctccggaagtagttgggcaagcccacctatatgtgttgggatggttggggaagccatccataccgtactgcctgatagggtccttgcgaggggatctgggagtctcgtagaccgtagcac 577 Hepaciviruscagggtttcgaccctggcccggatacctatcgccttacgccgaaaggtaacgagtaggagtcggg Itccccaggcccttaccgccaccaagccaggtggggaggtatgggagccggggggtgcagctggtagctccatgggggacgccccgtgagcggatgctgcatcgataccgggttagctctctgggagagcggcacttgacaccacgaatccgggaaccggacaatcgccggcgtgggacgcgttgcctccgtggccgagcaatttggcatgcccgtggtgaagagtgatggtgggggggggccccccttccagtaccgtactgcctgatagggtcttgcctcaagcccagagagtcgaggctgaaaaccgccatc 578Hepacivirusgccgctcccgaaagggagtccggcgcgtcatcccactccgaggagtggggtggcgtccccgtg Jtgccggggaaccatgaagcctaagggcatccacattttagaatgaacttgaagcacgtttcgctggccggaaagtcctgggttcccatggccagggttccgcaggtgggtaaatcccggtggggttccatccaggatatacggcaggcgggcgtagtccggcggttcggacgacgtgtgggtcgcctacggtggattgttcacaggatgggcactccggtgtgaataggccccgtcagggtgcgctgacgttaaactcaggccttgcctggtgttcggggaggattgcagggccacgccgcctctaagggccgtatggcacagtacttcttcgggcgggttgtcaaggccctccaacgcgacaccagtgcctcggcaggcatggggccacccagctcggcgtcccgcacacagacggcgtaggggaaaatcagcaatgtgaccccgggtggcattttccttctctctacttccatgcatgatcaaccgcaatc 579 Hepacivirusgggaacaatggtccgtccgcggaacgactctagccatgagtctagtacgagtgcgtgccacccat Ktagcacaaaaaccactgactgagccacacccctcccggaatcctgagtacaggacattcgctcggacgacgcatgagcctccatgccgagaaaattgggtatacccacgggtaaggggtggccacccagcgggaatctgggggctggtcactgactatggtacagcctgatagggtgctgccgcagcgtcagtggtatgcggctgttcatggaac 580 Icaviruscgaagtttaagctaagcaccctcgggcgttcccggattatgtgatcacatcaatttgatggctggtcaccacgcaacgcctggagagatactcttacttttctcttaagatccccggtcatttgacgcttgtaggatgatagggttattttccactataaatactttcatactcttggatgttctatatccaagacgggaggacctaccccgtaccccttagaggtgagatgccaagaacaggccctttctgttctctcgacaatggcatcataggcaacaagcatcacaccaagattgctaagttttgttaagagttcttcaagctatagggtggctgtagcgaccttctgatgcctgcggataccccttcggagcgatccgtgccacaggggccaaaagccacggctaacgcccatcaggagcggcacttaccccgtgccccacccttgaaacttgaatgttcacactggcttctctcggctttctgaactgtctgcagttggggccccgaaggatgccctggaggtaccccattttatgggatctgaccaggggacacctcagctctctaagttgctggtgtttaaaaaacgtctaagggcccccaccccttaggtggagggatccacctttcctttattttttaaaactcttttatggtcacaattgttt 581Antarcticctaggagactacgcagtgggataagatgactatgatgtcgtacgggcagaagccagtacagtcgapenguin agtcgagaccgacgtcgaggatttgactctgcctgacctagtgccatc virus A 582Foresttattggatccgcctccgggcaaaggttactttcttgtacctcggcttagccacagggtgaccccttgtpouchedacgtaggggccccgacgtagcactggtctgacaacaccttctcggcatttcaccttctgcccgctctgiant rattccgggcggtggtgtcaagaagcagcagtgctcttctcttttcttcctgcagttcaccgagccctacgarterivirus gggggtaggtg 583 AvisiviruscattcccctttccccagccatgggttaaatggcccctcaccaggttcggtgctgtctaggcttccagtPf-CHK1aaagaagtcaaccgagcattgaacaaaacctcagtgggtatggtagttaaccccgtccactggacaactttgtcctcttaaaagtggatcaatccaccccaactccccccctagccacctgagccatggtggatagcagtgacgaaactagggaccccaatacctctagtgccaagagaattcccccctcgcgagaggtgctcttgggcccgaaaggctagttggcagggtgaagtgaaggaagctgctagcgtggcaaccttaagcgtagcccgaagctgaccttagaggttaaccctagtggaccactggatgaagctgtggaggtggtggataggaaagttggccacttgtgagtagatgcccagaaggcataaggctgatctggggccagtgactataccgttccggtaaacctggtataaaaaccatgaaagcaagtgggtttaaaatttcttctaattccttcatttcagtagtgataactggcaga 584 Avianaccaaacaaggactagataacccacgtgaccgttaactggaaaataagatgttgtaggggcgaccparamyxovirustagttggaattcgaccccggctccgaaacctctaattgtggttattggcagtctagtctacttctaacgpenguin 585 Newcastleaccaaacagagaatctgtgaggtacgataaaaggcgaagaagcaatcgagatcgtacgggtaga diseaseaggtgtgaaccccgagcgcgaggccgaagctcgaacctgagggaaccttctaccgat virus 586Bat Hp-ttaagcttcggcttgttgcataggaccggaaaggtactatctaccctaactcttgtagttagactctctabetacoro-aacgaactttaaaactggttgtgtccttcagtagtctgtatggccattggaggcacaccggtaattatcnavirusaaatactaagaagattcatagtacatccttgtctagcttttggttggcagtgagcctacggtttcgtccgtgtcgctcacaattatccacacagtaggtttcgtccgctgtggttgagttgctagtccgttgctgtttcgtcagccatctacaactcgacacc 587 Basella albaggaatatggctaatcggcttattctaatcaaacgcaaaagacttatgacacagaccggacctgaacendonavirusgaggtgataaaacacctcgttcaggttcaaaacgtagaagattcattcctccgattagaaatacaactacgtctaagcacgatagagatggtatcaagatcggttttagaccacgagaaaatcggcaaatgaaagtacaagttgggtggtttaaattaccaagaacagtaacattcaagaacaacggcaacccgtttgttacctcatttcgtaaattgtttagaagtaacaaagataaattatttaatggtgggaagaacctaagtacagtaccagccagaagtagtgaaatgacagaaatgtttatgttcatgtccacgctagagggccaattgtcaatccaagatcgagatccaaaaataatcaataagtctatatacatgatagaggta 588 Ball pythonccccttcacccataggcactaggagaacaggataacccctaacggggcatcctgcctgtgacctttnidoviruscagattcgctagttagatatcttcacagactctgctaggcttctgacccagtccgttcccaaagtccgttaccgcccgagtagcgcttaggcgcgaaagggacggaagtacctccagtaagcgaaagctgaagtaagggaaatacggcaagactaacttgttagtcttacagtgtggataacctggtagttccccgatcccccaacgggcctccaaggaatcgagaaccaaccctcattccacgtctgtagtaagcaaaaacaggggcgatcttcaccgacacctctcaccacagagcacaccaacctctgtgaagccaatttcctcgtccaaggacaggttattgagggtcaactttcttccgaccagaagaagggatttcctaccaaaagaaaaaccaaatccaccaacaccacaaggtaaaacaacaacttgtgaagccaatacttagtcaaagactaactattgagggtcaactttctcttcaatagagaagggatttcctggtaaaacaaataacaacaactaacatcagcaact 589 Batgacaggtgttttggagggcggatgacgatatctggctggccaccagggaataacggcaaatgtctsapelovirusgatcatacggttcacaagtctaccggcgatagtggttcaacaccatgtgtagcagggattcttgcgtatgtgaaggcgacagtgc 590 BattaagcggaaagcattcttgtcccccggtcagtaacctataggctgttcccacggctgaaagggtgaPicornavirusacatccgttacccgcctcagtacttcgagaaacctagtacgcctgatgattccaaattggtatgatcc 3ggtcaaccccagaccagaaactgtggatgggggtcaccattcctagtatggcaacatacaggtgtccccgcgtgtgtcacaggcccttacgggtgccatttcggatgagtctggccgaagagtctattgagctactgttgatacctccggccccctgaatgcggctaatctcaaccccggagccactgggtggtgaaccaaccacttggtggtcgtaatgagcaattctgggacggaaccgactactttggggtgtccgtgtttcttttgttcatattaaactgttttatggtcacaacacaacttggtacgatttgtgattattcactgctcacttgtcacagtaaatatacacaatcatc 591 BatgggttttacgaaacccgtatacaccagaccttttctcccctccccctccacctaccttttccccctctttPicornavirusggaccgaaacaaggacacgtaagtggaaacgcgattttatatgtggttggccaccacggaataac 2ggcaattgtctacatgtgggaagtgcaacctccctgccgataacccctgaccgggtgtgtaggataggaaaggtgcccactgtgggcgacaggttatggtagagtggatacctagccaggggcaatgggactgctttgcatatccctaatgaagtattgagatttctctgctcattacccggtgatggttgtgtggggggggccccatacactagatccatactgcctgatagggtcgcggctggccgaccataacctgtatagtcagttgaattcagccaag 592 BatgaaacccgtatacaccggaccttttctcccctccctctccacttacctttttcccctcttcggcatgaaaPicornaviruscaaggattattcaagtggaaacgcgatttaatatgcggctggccaccgcggaataacggcaattgt 1gtatctgctggaagccaagcctgcctagccgatagcccttgaccgggtgtgtaggatagcccaggaaccagcaatacgcgacaggttatggtagagtagatacctagccaggggcaatgggactgcattgcatatccctaatgaaccattgagatactctggtcattacccggtgatggttactagaggggggcctctagtactagatctatactgcctgatagggtcgcggctggccgaccatgacctgtatagtcagttgatttgagcaat 593 BatacgaatcggtatacgcttcggtacctattgagcaagttcgaccctattacgatttgcctgcccgaattIflavirustgactcaaacaattgtgacatactatgtctctgatgaaagcactacacgagtagcgcccagcatgtatgttttcaagtatagtataagtctgcctctatagtggattattgaccttaaagccagtcaaccatcctatatgctgcatcgagacttgatgtcaatctgcctctactacgcaaatgtctagtaattagttataaggttttactattaccctcataccaatatagtagtagtgtatgtgagtatcattcttactccgactgttaagagaaaccaatttatagtcgttaaatatgataaatggaatgaatgatggtgtcatataaaaacactcactctataggcgtaagcattctcgctcttagagtcgtaaagaagaaatgccgtgtctatcagtatgttatgcgatttattttctgccacgcgatctagtgcaatctagttgacatacagacattgcctaccactcgcgagggtcgaccggtagtgtaaggagtaagtgatgataccgcttattctgtaccattgcctggtgaggacagatcctgactaatataaatataaatgaacactagatccaag 594 Batgtatagcaccggaatggtatatactactccaagtatacgtactaggagttaaaccctgtaatttacagdicibavirusgggatttagtgactatatccgtaaaagtcgattggacgttaatcggtaacgaggccaagtaccgtgaaccaatttaaaaacgtattactcatgtggtagaaccaacttggaaatagcatggcatataggagat aggg595 Betacoro-gataaagtgtgaatcgatccgtagcatcgcaccctcgatctatgttagatctaatctaatctaaacttnavirustataaaaacactaggtccctgctagcctatgcctgagggataggcgttgcatactagtgtcttaggaHKU24atttgactgataacacttccctgctaacggcgtgagcactctcagtctaagcctcccacccataggaggtatc 596 Betacoro-atttaagtgaatagcaggctatctcacttcccctcgttctcttgcagaactttgatataacgaacttaaanavirustaaaagccctgagatagcgtattgagcacttgtctggtgggattgtggcattaatttgcctgctcatcEngland 1taggcagtggacatatgctcaacactgggtataattctaattgaatactattatcagttagagcgtcgtgtctcttgtacgtctcggtcacaatacacggatcgtccggtgcgtggcaattcggggcacatc 597Boonetacgatcgctgtacattccactactgccaattagctccccatcccgttgctcccctctataaggagacardiovirusgccactcttgcaaaggtgaagccttcacccccggtcgaagccgcttggaataagacagggttattt 1tctcctctcctcggcgcttgcctcactaagctgaataggactatctattcaggcggatggtctggtccgttccacttggacagagtgtgtatctgggattccggatctcgaccacacactcaccagagctcaggagtgattaagtcaaggcccgatctgcggcgaaaaggaaatgaagtattttgcagctgtagcgacctctcaaggccagcggataccccacctggtgacaggtgcctctggggccaaaagccacgtgttaatagcacccttgagagcggtggtaccccaccaccctgcaaattatggatttgacttagtaactaaaagattgacttggcatacctcaacctgagcggcggctaaggatgccctgaaggtacccgtgttgaaatcgcttcggcgaccatggatctgatcaggggccctgcctggagtggttctatcccacacagcgtagggttaaaaaacgtctaaccgccccacaaagaccccggcagggatgccggtaccatttaccaattcttg acact598 Breda virusatcacctagtacttacaagcgggtcaaaccgccctccggaacggtcataaccccctcccgaacgtgcgcttgacgtgactggtattcagtctagctactgagaaatactccggggttgtaacccaccatatgacctttggtcagtaggtaacactccaaccaaacagcatctgacccacctccagcttgctgcaggccatttggacCaaacgggttcagatatcagtggctaaacctctgacccacctccagatactgcaggccttttggactaaacgggtAcagactctagtggttagtattaactacccactgatagccgccaacctgatttttattgttacaaaattagtgatacacattattacttacggaggcagtagtaggagatgcacagtttttgctgataccaattatactgtgatttggtgattcggctaaggctgatttcacatacttagatgcttgaagtaaccacacaacatctgattgataggtactaaggtaaagagatcaggaaaaaacataggcgcccatcagtggtgtctagattaattaatctggcaaacaagtatcaagtcatcgactccctaggagtgagacttacgagtaccaattcgcctattaggccatccatataaaa 599 Bovinegtatacgcccagttagttcaggtggacgtgtacgattgggtatcccaaattaataataggatagggaviral ctaaatcccctggcgaaggccgaaacaggttaaccataccatagtaggacgagcataatgggggdiarrheaactagtggtggcagtgagctccctggatcaccgaagccccgagtacggggtagtcgtcaatggttvirus 3cgacgcatcaaggaatgcctcgagatgccatgtggacgagggcgtgcccacggtgtatcttaacccaggcgggggccgcttgggtgaaatagggagttatacaagccatgggagtacagcctgatagggtgttgcagagacctgctacaccactagtataaaaactctgctgtacatggcac 600 Bovinettttgcggctctgccgccgttcgggattacctgattcacagagcaaaacaggacctctagatcgtgrhinitis Acttaaacgagatcatgctcgaactagaactataacgctggtcactggacccgtgccgcgccttgcg virusgatctttgcgggaatggtggctagtgggctgtggaagtgactctaaccacacgcccctcaagtgtgggaaaacacgaactggtgtagcgacgacgataggccagggacaccctctccagtgatggagacccaaggggccaaaagccacgccttgtgccctgtcgttcacaaccccagtgcagttcgtgccagtacctgcttttgggaagtgtgctaggacagctgaaaacagtcctagtgggagactaaggatgcccaggaggtacccggaggtaacaagtgacactctggatctgacttggggagagcgggtctgctttacagacgccactctttaaaaaacttctatgtctcgtcaggcaccggaggccgggccattcattaaaacaatacacttt 601 Bovinettggatctgagcaggggccccctagggttgattacaactcaactgggggttaaaaaacgtctaacpicornavirusccgacacgccagagggatctggtaccattatttcattactcaccactggatgcagattgacgataaisolate TCH6acgttgttgtttgtgactattgacttgatctgatctacgggatactacactgttatacttcttgctttgatggtgttcactgtactttgtctccttctacatttcaca 602 Bovinecaccaatagattagtcaagctgtctataggcataaactaacccccaaccccattaccccggggccanidovirusggtgggccgccgccttcgggcaaacccgtgcgctggtataatcaaggttcacagccagattcact TCH5gccggttagctagtggggcggtagcctggcaaaacccgaagaggttggaaagggaacttcagggtagtttatcctaggctagcgtagctacagttcggtcaagataaccgtcctggtgctagggctagtagagacagtggtaacttggacaagggtccagggccactttagggaataccctacggaaggctaggtccgtaaggaagacccccgcagagtccgcggttgagcagagctcctgcgtagacaaaaggcaaaaagtggattacattcgcctgcaggaaaaggcaaacgtcgaggagtcggagctaaagtactggacgattgataccacgcctgctgcggtagataaa 603 Bovineccatcgacactccaggctcacggattaagttaggttccgccgaagcgggctaaccaggcccctaghepacivirustaggaggcgcctatcccgtgagccattccccacggattgagtggagctggagctgggaaggaccgagtacggtccaatcgagaagaaccctgatgaacattccaggcctatcggtagataggatatatccaccagtgaaggcggggtcgtgggtacaggccccctagtccacacagcctgatagggtcctgccgcaggatccgtgggtgcggctgtacatgtacc 604 Botrytisgccccgccgaccacctatttatactaaataggaaggtcccgactagtcggataattcggataacgcinereaaattatggttagatctattaaagttaaaatagattgaatactactcatcctccttattcctatactagggamitovirusgtaaatgacaaatgtctatcctcaaaccgaaatggcttaagtgatgaatttgaaagaaaggtaggat4 RdRptaagaatataaggcatcaatatattatacccttgaatgttaagtgaccacggcgtgacgattagggctatcttaggatagacagccatctaacgcgacagcagtggaaatcagcttagcatctcaagatcatgtataatatatacataaccttacaattataaaaccaaaccaaaacacactataatatatataaattatagaagtatcggacctggacggtacctactattaactgatagtagccaaatgcaggaagctc 605 Botrytisggaacttttcagttccagaagtggattattaagcatcaaagatacactttgaacctgcgattaattcccinereaccatagtgactcttgttactgtgaattaggaatagagtagttcaacttctaatgaggtgaacaatataamitovirustaactcatcttattaaccctatacgtagacaattgtccaaagagacagaggaattctgccaatctgga2 RdRpatgtttggtacgcgtagaagataataagagaccctctattcccctgcctcatgactaagtcatggcccggggtgtaatagagatacttttatatattatacaatc 606 Caninecgctctttatacaaatctgtcaaccctagtataactctaagccgaacaattatagctaggctattattatpicodicis-ataacattaattaggcattagcgagtcgccaatctcaggtaatcctaaggataccatcctgagactatrovirusagatgaagcgccttcggttaccgatgcccggtgtccacgaagccatcgtggtcggccgcgtccccstrain 209cacctctcccaacttggactccatgattcagtaggtgtaatgattagtattattgattctgctcgttcaatgtgtttatcttcacgatctgggacccaacacatgcttcactcatgataaatgaggaccctcattagaagacacccaaaccatagagtgcgagaatgaggatactacttccattctggtaacagaaatgaattcctgcgtgtgtctcgtaaatggaatattaagaacttcagataaatcgaacaatacactaatacaagagttttctaccaacatgacaatgcggctaatctgaccgtggagctgtgaagcgctcaaacccgagtgagtatacagtcgtaatgcgtaagtccatgaggaaccgactactgttacctctgaggtgtgtactcattCCtctcttttattattatttgttattgcaaatactacaactttgatcaac 607 Canineaccagacaaagaggctaaggatagttaaattattgaatatatattaaaaacttagggtcaatgatcctdistemper accttaaagaacaaggctagggttcagacctaccaat virus 608 Caninetttaagtgagtgcccaatctcttgactcctgctggaaccaccgaccagtagtgtccaaaatgccaggkobuvirustggaaaatcctccatcccctctgggcttcatgcccggcatcctccccccagcctgacgtgccacaggctgtgcaaagaccccgcgaaagctgccaaaagtggcaattgtgggtcccccctagtcaaggcgtcgagtctttctcccttaaggctagtcctgtcagtgaactctgtcgggcaactagtgacgccactgcatgcctccgacctcggccgcggagtgctgccccccaagtcatgcccctgaccacaagagtgctgtctggcaaacattgtctgtgagaatgaccgctgtggctgccaagcctggtaacaggctgccccagtgtgcgtaaactcatccagacacggtctggcaacagctgaaagacatggcgtaaggggcgtgtgccaacgccctggaacgagtgtccactctaataccccgaggaatgctacgcaggtacccctggctcgccagggatctgagcgtaggctaattgtctaagggtattacatacccaccctcacacttgacata 609Camelcttaagtgtcaatctatctatagatagaaaagtcgctattagactagtgtctactcactcaactaaacalphacoro-gaaatttttgctacggccggcatctctgatgctggagtcgtggcgtaattgaaatttcatttgggttgcnavirusaacagtttggaaataagtgctgtgcgtcctagtctaagggactgtgttctgtcacgggattccattctacaaacgccaactcgaggactgtctcgtgatgtgtggaagcaaagactgtcatgtggaaaccagtaactgttccta 610 Cripavirusgcaaaatcggtagtacgaaaacgtacgaccaccgatgagactgaaatgacactagagagattatttcaatatcctagtgaataaagtcaatatagaggagatcgatcgtcaatcgatggcgctgacagccggaaagacggcaataataaaaaccaagatttagtattaagattgattgaattgcaaaagctatcagaatagacaatcaaaatattaagtaaagcaaaagcacttaaagaagacaatataaattagttagtaaccaaacctcatcgtgcccctaagggaaaccggaacgtaaaagcgtagaggtattaaggtcactgcggagacctaaaatccgcaatatatgattgtaatgattagaatagacttagatgtaactataagagatataaatacttgtttcaagatttatagacaagatctgatcctatggattttagataaccttcatgttagtggatagtgtgtgtacctatctaaacgcataaggctcaatacatatttaaagtaggactatgtattacggcgcatctaacggtaacgttagtcaagaccggagaatctcggaatgaatatagtaaacccaaatttata 611Human acctagtgcgcctgattatatccccaccccgagtaaacgttagaagttacgcaaccccgatcaatacoxsackie-gtaggtgtagcactccagctgcatcgagatcaagcacactgtctccccggaccgagtatcaatag virusactgctaacgcggagaaggagaaaacgacgttacccggccaattacacgagaagcccagtagt A2gccgtgaaagagcggagtgatcgctcagcacacccccgtgtagatcaggctgatgagtcaccgcgatccccacaggtgactgtggcggtggctgcgttggcggcctgcctatggggcaacccataggacgctctaatacagacatggtgcgaagagcctattgagctaaaggtagtcctccggcccctgaatgcggctaatcctaactgcggagcacatgccctcaaaccagggggtggtgtgtcgtaacgggtaactctgcagcggaaccgactactagggtgtccgtgatctattattcaataatggctgcttatggtgacaattaaagaattgaaccatatagctaaggattggccatccggtgactaacaaatcgctcatataccagtttgttggttttgttcccttatcacatacagctcataacaccctcaatatttactacaattgaatagcaagaa 612 CoronavirusagaaacaagtagtgttttaaaaaccacaaattagtgcctgtaacatcatgcaatgaaagtagcgctcAcCoV-JC34actagcctctatgcaaagaatgaaaaagaaatacgaagcatttaaagaatacaatctatctaggataggtacaaactcctccccctcagacacggtcaactcaactcaactaaacgaaatcccccagcatggttccgacccgtgtaaggagtgtatacgtgcagtcgagcccttactagtgtaagcgtaacggcatctaggtttgcacgtcaggaggaaacggtgtgtacgtactagtgatacgccgtatcggttccggcccgataggtattgcattagacgtcctgggtggactgcctgcccttgtgtgattcggctgaccgtcagtttggtcacctcacacgtccttaagac 613 Chickengggtatggtggaaaccccgtccactgggcatcatgccctctcagaagtggatcaatccaccccaapicornavirusctccccccctggaacctgagccacagtggactccggtgacgaagctagggaccccaatacctca 3agtgccaagagagtccccccctcgcgagaggtgctcagggcccaaaaggctagttggcagagtgaagtgaaggaagctgctaacgtggtgaccttaagcgtaattcgaagctgacctttgaggttaaccctagtggaccactggaggaatctgtggaggtggtggttaggaaagttggccacttgtgagtagatgcccagaaggcataaggctgatctggggccagtgactataccgaccggtaaacctggtataaaaaccatgaaagcaagtgggtgaaattactctattatccacattcagcagagatattggcaaa 614 Chickengtggccgacagcagaacacctaccgaaccaccacctcacccccataactccaccctctacacctpicornavirustccgctatggtggaaccactgcatattgccagactgagaatggccaccccctcgacacctgcccc 1ctactgccccaccgcgcaaccattgcagtccactcggaggagaggcatgggggccccgacatatccccagtccagaggtgaccaccccctccccgtccggtagatggtccagagggctagccgacgccctctatgatgcagcagtctacctccgtcagcgcgagcatgcacagtcgagcccacggaaacacagcctagcattgcactctcaccctgcgtaccctgggcgccaccgctcgagattcgcatgacgacaccctggcgtccccccaccgctacgtgatatactcgtggcatacaccgccctggcgacagtacattccactgcctaatagggtggcctccctcaatctcccgcaccccccattgcgcacgtcatcaccgccgccgctaacgcgatccggcgcggactcactggcactgtcccctcgtccgccgggtaccactcatggttggcttttcacttaactggaactggtgaccatcctacacatgatggtcgccatgaccat gac615 Chickenaaaccctcacgagtgcagtggtaggtcccaggccaatattcacgtaaggcaggaccaattaccaorivirusccactcgtgtagggactggcctatggtacccagaggggcggtagggggaattaactccccctcc 1cctgtggtcctataccaccccacacctctgtgggcatcatactatcacttgattccgactataaacactaggcaggcgcgcctagtcatacaccgcccggctggtctaccagctcagtgggcggtgcgcgctggtccatcgtgcccagcgacatagcaccagtggacacctccgaacgccctcccctgtatggggtggtgcccaggggatcagtgtggtgacacactccctggggcccgaaaggctagtgtgcaacaggtgaggtacagccagctgcccccgtggctggagggaccaagcagtgaagcacacctcaccacttgggggtgggctagtaagtggtgaaagcatagtgtccgtgtcgctggccaacactagggtcaagtccagccactcagtgagtagatgcccaggaggtacccctagtggatctgacaggggcctgttacttaatgcaggttaaaaactatgaaagctgagtagtgtagcccggctggtggcactcaccaattcattctatttt 616 Chickenggttaacttgataaccaaggcaccgtgcagggcagcacgttaggagacatgagattcatgctccgallivirusaatgcccaaaactagtgtgatgatatgtctacccaaagtacccccaaggatcggtactcaaacct 1taattcctagtcccatcattgggccaagtatctaggaaatgtacccgtgccagacgaacgtaagaaagctgtcttttattgaacggactaatgaactaagtataactggctcgcgccacctggtgtgtgccgaggaattcccccatggtaacatggtccaacgggcccgaaaggctagtgggcaatcggtcctccaaggaaggggacccaccccgacctgaacaggatagatgaagctcacctcccaggctcctaaccccaaggaagttttacttatagtaattagaaatagtatgtaattgctggcaatcagctagtagtcaggaacgttatgaccaaatgagtagacccccagaaggtaccccattatatgggatctgatctgggcctcatactgtgtgtctccccacatatgaggaaaaaccatgaaagtaggtccaaaatattataccattatcattctttagtggtgacgccattatatcagcagtagctg 617 Chickenggtgcatcatcactgaacaccctcgggcagagatgcaagggtggaagtcactcctgccccctggcaliciviruscaacatgcaggtgcccgatcccaagcttagactgacacctctcctgggtggtgcaacactccaaggttgatgaacaaacctggagggacctctgggcaacctggtctctcgaggatctccggcgcatctccacagactacctcatgctcccggaacctgagaagaacactgatgcctatgatggctggctgatggtcggcgagatgagaccatgccggtctcattggctgggagg 618 Carpagctacaggaaagagagagataatcacagcacataaatacaactacagaagagagccatccctgpicornavirusagcactatttacagcaaaccacgctgggaaaagtggtagcatgacccacttacgggaacttagtat 1aggattttaatatcttcgattcatattactatcaaactaagacgcccggacatatccgtgattatatagtttaagcaaaattgagtaactaagtattaacctgccaaatggtgagaagtaactctgtgaaaataccatttgtgcatgaaattgtcagaaaactcaaggcattggggggtcccactgctgatggaggactattgacagactctattgtagagagtagtgactaatgataacgatagcgtattacgcaatgggctgtacccgttagatttagtatgccggggggaggggtcccactggattgcactatgtaacctgacagggcgtctgccgacgcactacaatgaggataagatcggctgatttata 619 Falconcgcttggaataagagagaggaattatgcatgctagagtgatgatacaactaattgactaatccaapicornavirusgtgaagctcacgcaggggcggcacgacacagccgtaattcactaccgtccctccacaccagtggatgaagggccggatgtgtggcctctggctaacccctctctctggggtgatgctactggatgattactcctagaccaaatcacatgaactcctcagatccacacggtggggctatgagcctgcggattaatagctggcgacagctaccccaggggccaaaagccacggtgaagcagcaccctcatagtctgatgcccaagggctgatgttgggagctagtagtgtgtgtctggcctatgataggacactggccaagcgcagaggagtggggctgaaggatgcccagaaggtacccgtaggtaaccttaagagactatggatctgatctggggccccctcacgtggcataccacgtgagggggaaaaaaacgtctaggccccaccagcccacgggagtgggctacccaaaaaagcccaacaatatttatggtgacaattcactgatcactagcaatttttgtattcaggactccaattattgatgcagatatagtggacattcagattcaaatacaaa 620Equine cgacaggcacaggtcgctccgagactagtagtgtgggaacttgaactactgatgaaacgaggtarhinitis Bgtgacactcagtacctgcgaacgaggtcggggccctcccacaccacacccaactacactatcgt virus 1tccactttagcaggggtcactactatccccctggcggcattggaactagccgtcgcgtcaaacgcgcagccctgaaggccccacaccagtggatcagccgtgggtatgatctggcatgtgatctcaagcctgcaaccgaagccgaacagccacatgaacagatgagcgtggtagcgctgtgtgagttggcggtggatccccctcgtggtaacacgagcccccgtggccaaaagcccagtgatacagcacctctcacatccaggacgaccccatcctggcgctcactcaagtagtatggcttagtacgcattaggtggtaagccgagactccctcggccagactgaatgcacacatgtctaggggctaaggatgtcctacaggtacccgcacgtaaccttcagagagtgcggatctgagtaggagaccgtggtgcactgcatacagatgcagcccggataaaaagcgtctatgcccctacagggtagcggtgggccgcgcccatccattaaaactacttgttct 621 Equineaagggaactgctcgtaatgagagcacatgacattagccaagatacctggcaattgtcacgggagrhinitis Aagaggagcccgactcgggcacttactctcaaacaatgaggcgcgcctcggcgcgcccccccat virusttcagccccctgtcattgactggtcgaaggcgctcgcaataagactggtcgagcaggcttactattgtttcaggcatagcgcgcccagcgcggcgggccgtcaagcccgtgtgctgtacagcaccaggtaaccggacagcggcagctggattacccggtgccattgctctggatggtgtcaccaagctggcagatgcggagtgaaccaacgaagcgacacacctgtggtagcgctgcccagaagggagcggagctcccccgccgcgaggcggtcctctctggccaaaagcccagcgttaatagcgccactgggatgcaggaaccccacctgccaggtgtgaagtggactaagtggatctccaataggcctgttctgaactacaccatctactgctgtgaagaatgtcctgaaggcaagctggaacagccctgatcaggagccccgctcgtgactctcgatcgacgcggggtcaaaaactgtctaagcagcagcagaaacgcgggagcgatcatttcctcatttgtttc 622 Equinegctcgaagtgtgtatggtgccatatacggctcaccaccatatacactgcaagaattactattcagtgarteritisggcccctctcggtaaatcctagagggctacctctcgttattgcgagattcgtcgttagataacggcavirusagttccctttcttactatcctattttcatcttgtggcttgacgggtcactgccatcgtcgtcgatctctatcaactacccttgcgact 623 Enterovirusactctggtatcacggtaccatgcacgcctatatataccccaccccatcgtaacttagaagcaacaa sp.acaaactgcccaatagcagcacaacacccagagtgaaggggcaagcacactgatccccggaa isolategggtctgacggtatgctgtacccacggcagaagtatgacctaccgttaaccggccatgtacttcga CPMLgaagcctagtaccattatgaaggagattgatgaacgctccccagcaaccccagctggtagactggtcgatgagtctcggcattccccacgggcgaccgtggccgaggctgcgaggcggccagcctacaccatacggtgtaggacgtcaagatactgacatggtgtgaagagcctattgagctacgtggtagtcctccggcccctgaatgcggctaatcctaactccggagcatccgccagtaagcccactggaagggtgtcgtaatgcgaaagtctggagcggaaccgactactagggtgtccgtgtacctgattacttattgatggctgcttatggtgacaacttatagttatcatcataagctacaggtcagccaaccggagaattataggttatttgttggtttcataaacctacagtcgtattacctgtcaattaattgactcaaaattaacaaca624 Enterovirustaccgctgcaccagtgagctggtacgctagtaccacgcacggagtagatggcatcccccacccc AN12gtaacttagaagcaaagtacacatctggccaatagtggcgctgcatccagccgcgcaacggtcaagcacttctgtttccccggtccgcaagggtcgttatccgcccagtccactacggaaagcctactaaccattgaagctatcgagaggagcgctcggccacgaccccggtggtagctctgagtgatggggctcgcaaacacccccgtggtaacacggatgcagcccgcgcgtgcactcgggacagcctattggttgttcacctcaacatagtgtaaatggccaagagcctactgtgctggattggattcctccggagccgtgaatgctgctaatcccaacctccgagcgtgtgcgcacaatccagtgagctacgtcgtaacgcgtaagaggaggcggaacagactactacggtactccgtgatcattgattatatgaatatatggtgacaattgctgagatagcgaattagcgactctaccgctgaacattgccctgtactacctaatcgcatacacaaaacctcagagataccaagctcaacattgatctgcagattcctgaatctcaaatataaattggaacaagc aaa625 Dolphinaccagacaaagctggctaggggtagaataacagataatgataaattatcatacttaggattaatgatmorbillivirus cctatcaaaggcacaggataggataaaggacacagtc 626 Dianketgttttcaaccataatactactactacaagtataaaaccccgtccgtctgtcggagacgctaaactctgvirus accaccaatctagccacatcagagcttaaagaacctcagagacactctcccacttaacatcattaggaatcttcgatgctacaacaacaggctagtgaacaataaatccgtacaattcacagagtaagaggccataggtccagactagaaaggtagtactattgaacaaatacttagattaacagaggctataaatagtgctcatcacgaaacagagtaaccagtgcaatagtatgagcagagtaaaacgtcagatacgac acc 627Guereza cactcaatactacactccgcataggggagaagcgctggcgacgcggaaccgcgttaaccatachepacivirusgcgtagtacgagtgcgacagaccccggtgctactggtggtagcgagacacgagccgaagtctgtggggggaactccacttagagggcatgcccgggcgtaggcttctgagagggatgggccccaacttggcccctgagtgggggggtgttacgacctgatagggtgcgggctggcgcctaccactaccagtcgtacatgagtc 628 Grapevinegcccggggggtgcagtcctgtgaaagggtctgcaccatactatatatgtatatgattacatcccaaaassociatedaggcgacttcgttcaggattaaatctgacgtaggtccagtaaataagcatgtcaaaacatgtaagatnarnavirus-1atcctgtaatctactctcataagatgagataagatgatattgcagacccatgtaaataaatccattatgaattcattcatataaggtagaagtggtaactatggagaaacattaatataaaacggtcattagcatgaacgtcattaaggaactggcataccaatgtctatttagtgactatgatatttagagtatcccttatattaattaacaattattccattagcatatcatccgacaacaaatataaaagaagaaatattactcattaaaa 629Goat gtacttacaagcgggttaaaccgccctccggaacggttacaaccccctcccgaacgtgcgcttgatoroviruscgtgactggactcagtctggctactgagaaatactccagggagtatcccaccatcttgacctctggtcattaggtaacaccataaccaaacaaactctacacacctaacccacctccagcttgctgcaggccttttggactaaacgggataggtgattgtgaccaactcgtctacccacctccagattactgcaggcctttttggactaaacggCatagacttagtggttagtattaactacccactgatagccgccaacctgattttcattgagtaaaattagtgatatacactattacatacggaggcagtagtaggagatgcacagtttttgagataccaattatactgtgatttagtgtattctgctaaggctgtatatacatacttagtaggagaagcaattatacaacatttatattgatttgatactaaggtaaagagtcttaggaaacaccatagacgccattcttgtggtgtctagaccaactaatctggcaaacaagtaccaagtcattgactcactaggagtgagacttacgagtaccaatttgcctatttcggacatccatataaag 630 Foot-and-acaagcttgacaccgcctgtcccggcgttaaagggaagtaaccacaagcttacaaccgcctaccc mouthcggtgttaatgggatgtaaccacaagatacaccttcacccggaagtaaaacggcaaattcacacadiseasegttttgcccgtattcatgagaaacgggacgtctgcgcacgaaacgcgccgtcgcttgaggaggacvirus Ottgtacaaacacgatctaaacaggtaccccaactgacatacaccgtgcaatttgaaactccgcctgisolategtctttccaggtctagaggggtaacactagtactgtgcttgactccacgctcggtccactggcgagtgttagtaacagcactggtgcttcgtagcggagcatggtggccgtgggaactcctccttggtaacaaggacccacggggccgaaagccacgtcctgacggacccaccatgtgtgcaaccccagcacggcaacttttctgtgaaactcactctaaggtgacactgatactggtattcaagtactggtgacaggctaaggatgcccttcaggtaccccgaggtaacacgcgacactcgggatctgagaaggggactggggcttctgtaaaagcgcccagataaaaagatctatgcctggataggtgaccggaggccggcgccatccattataactactgacttt 631 Felineacttttaaagtaaagtgagtgtagcgtggctataactatcattactttaactagccagtgctagatttginfectioustcttcggacaccaactcgaactaaacgaaatatagtctctctatgaaaccatagaagacaagcgagperitonitisattatttcaccagtaggcaatcactcctaggaacggggagagagaacggcgcaccagggaccg virustccctgtaggtaagtcgtctagtattagctgcggcggaccgcccgtcgtagttgggtagaccgggttccgtcctgtgatctccctcgccggccgccaggaga 632 Farmingtonacgacgcataagcagagaaacataagagactatgacatagtcaccctgtattcattattgactatatvirus gacctattattagaccatcacgggtaaatccactcatgcagactcgccaagtacctccaaagtcagaacg 633 Avianacttaagatagatattaatatatatctattacactagccagcgctagattataacttaacaaaacggacinfectiousttaaatacctacagctggtcctcataggtgaccattgcagtgcactttagtgccctggatggcacctgbronchitisgccacctgtcaggtattgttattaaaatcttattgagctggtatcactgcttgattgccgtgtctcactttvirus atacatctgagcagggctacctagtgtccagcgtcctacgggcgtcgtggctggttcgagtgcgaggaacctctggttcatctagcggtaggcgggtgtgtggaagtagcacttcagacgtaccggttctgttgtgtgaaatacggggtcacctccccccacatacctctaagggcattgagcctagcgagggctacgttctcgcataaggtcggctatacgacgtagtagggggtagtgccaaacaacccctgaggtgacaggttctggtggtgatagtgagcagacatacaatagacagtgacaac 634 Humanttaaaactgggtgtgggttgacccacccacaccacccaatgggtgagtactctgttattccggtaacrhinovirustttgtacgccagtattccctcccctccccatccattacgtaacttagaagattaaatacaagaccaat 1agtaggcaactctccaggagtctaaggtcaagcacttctgatccccggttgatgagatatgctccaacagggcaaaaacaacagataccgttatccgcaaagtgcctacacagagcttagtaggattctgaaagatctaggaggtcgttcagctgcatacccagcagtagaccagcagatgaggctggacattccccactggtaacagtggtccagcctgcgtggctgcctgcgcacctctcatgaggtgtgaagccaaagatcggacagggtgtgaagagccgcgtgtgctcactttgagtcctccggcccctgaatgcggctaaccttaaacctgcagccatggctcataagccaatgagatatggtcgtaacgagtaattgcgggatgggaccgactactagggtgtccgtgatcactattcattattaattgcttatggtgacaatatatatattgatatatattggcatc 635 EV22ccttataacccgacttgctgagatctataggaaaaaaccattcccagccaggggtggctggtcaataaaaacccccatagtaaccaacacctaagacaatttgatcaaccctatgcctggtccccactattcgaaggcaacttgcaataagaagagtggaacaaggatgcttaaagcatagtgtaaatgatatactaacctgtattatgtacagggtggcagatggcgtgccataaatctattagtgggataccacgcttgtggaccttatgcccacacagccatcctctagtaagtagtaaaatgtctggtgagatgtgggaacttattggaaacaacaatttgcttaatagcatcctagtgccagcggaacaacatctggtaacagatgcctctggggccaaaagccaaggatgacagacccattaggattggatcaaaacctgaattgagtggaagatattcagtacctatcaatctggtagtggtgcaaacactagagtaaggcccacgaaggatgcccagaaggtacccgcaggtaacaagagacactgtggatctgatctggggccaactacctctatcaggtgagttagttaaaaaacgtctagtgggccaaacccaggggggatccctggtaccattattgttaatattgaca tt636 HumantccgacgtggaggaattaacatcattccgacgaaagtgctattatgcctccccgattgtgtgatgcttTMEV- tctgccctgctgggcggagcgtcctcgggttgagaaaccttgaatcattcctaggagccttggctclike ccccggtctaagccgcaggaatatgacagggttattaccaaactattatactactacatgggactcardiovirusatccatgaaaagggtatgtgagcccatccactaggagaatctgcgcggcggtctttccgtctctcaacaggcgtggatgcaacatgccggaaacggtgaagaaaacagattctgtggaaatttagagtggacatcgaaacagctgtagcgacctcacagtagcagcggattcccctcaggcgacaagagcctctgcggccaaaagccccgtggataagatccactgctgtgagcggtgcaaccccagcaccctggttcgatggccattctctatggaaccagaaaatggattctcaagccctccggtagagaagccaagaatgtcctgaaggtaccccgcgcgcgggatctgatcaggagaccaattggcagtgattacgctgccactttggtttaaaaactgtcacagatctccaaaccaagtggtcaggattccaattagttgactgacaat 637Humanacttaagtaccttatctatctacagatagaaaagagctattagactagtgtctactatctcaactaaaccoronavirusgaaatttttgctatggccggcatattgatgctggagtcgtagtgtaattgaaatttcatttgggttgca229E acagtttggaagcaagtgctgtgtgtcctagtctaagggatcgtgaccgtcacgagattccattctacaaacgccttactcgaggaccgtctcgtgatgtgtggaagcaaagactgtattgtggaaaccagtaactgttccta 638 Hubeizha-gtgcaggatggcctttcccatcttaagtggtagtaggatttcgtgggtccataccccccgatttcttgovirus-gtacgtattccatgcacggagaatacgaccaaaactcttatttcaaaaaatattattattactcagtgglike virus 1gctgagtgcgacccaccagaccagcttagcaacctggaagttgaggagatttatggaaccaaattacacatgcgtggagtgccgccactccgtatctgacactcattacgcgattaagactgcgacgagacgagcgaa 639 Hubeitombus-ggaccatccaggcaggtgtaggctagtaccctcacctgacctgtcgcgatgatggctttgtgagglike virus 9cttgtgggaggatcccaggcccatgcattgctgctgtcatcgtgaaaaatgagtatgctcgcacctggcgtggaggaaacggcatttgacggatgctaaggaggatttgaaggtcctcaactcccatgccttattacaagtcccattcatcaagcttcgagcccacgtctgtgacgagcagaggtgaggaggagtcaagaggagtcctattcaagctgacgcagcaagaaccataaaactacattgctcaatgggcaagagcggctaaggctcggactcgtagcacgacagtgtgagcggagccctgtaaatgtcgcggcccttcatcggtggatgcatcggagataagaaaattggcatgaacttgctccaggatcgtacgtgatcgatgaggagttgaacttagtaggaaccaacgtatgagcgtatggtgtccgaaactaagaagcaattccgtcatcgggcacgtatggaatactacaacgagaaaaaatgccttgaaaagatccactaggaacgc 640Hubeitombus-ttcgggatacccgcgtaagcggccacactgactggagtcggtgagaatttgtataccagatgagglike virus agacgttaccaccgtctcggcagtgctacgtctgggaaaggactgtgatagtggacagtcctacc 32gtctttagatacattgcgagtgtatagcccgggagggattaactaatagcaacgcaatgcacacggcggttcggatttgcttgactgatggaaagacatctaagactaattgaacc 641 Hubeisobemo-gagatgatgcgtgggccgttgcgctgcgggcggcccacctccctacggggaccgtgagacacclike virus 3gctggggaaggcccccacccccggccaaggggatcctgccgagaggcaggagaaagaggcccagccctctggggcgcattaggggTgcctgggagggaagtacccgagccgggcggccggtcgggtgcggctgtgcagttcgaggctaaccgtaaggaaggcctgagctgcctcggcttgtcggaaaggaagacgaaggcacttatcaaggattcaggaagcaggagcgcaattacagcgcgcgccgtgcggcctggattaaccgcatttggccg 642 Hubeipicorna-agcaacttctactgaaaactagctagagttcgacgatctctctggctaatgacaaataaccaatcaalike virus 2aaagtcaaatgttcatgtatatatatatttagtagtgacctttatttagaaaaactttagatgatttatcgtcaagttgccctagtgaagcgatcagctattatatcgatcatttagtatacgtcttaattgacgagtaagtacgttttgcatacctcacattgaggatagtatcgtacctgactaagaagttaaactagtctaccaatagcaaccatataggatatagattgataaacaaggataatctgatcaatgctcagcttacggtgattatgtatagaaaaGtattataaaaactacataattgtcaaaagaaaaagcgttacgtactgacgcataatgttcacagtgtgacacaaaccactatattagattgtaaaataggctagcctgaccatttatcaaatacaaactgatcaaacgcctctccgagccataaggccgacgcgaatcgacataacagggtgagatacagctgcagagcagccgaggatccacttatagagagagaattactcaaacgaaattagaagtcacttctacacagaaagtctgagtaggcgttatccaaacgtaaa 643 Hepacivirusacatgggggggggctgacagtgagtacactgtgccaagcaggtgctacgctatgcctaggtgct Pgctgtaggccaaggacatgtcccagtcatcccaggtgagggggggggacccctcaccgctgccactgcctgatagggtcctgccggagggtctcggtgtccggctgtac 644 Harriergatgtgtgacggtgtaattactttccggatcccactttcctattataactctttcatcccaaggttagggpicornavirusaaagaacctggctcggtaccaccagaccctccgccacgctagtggactctccggagataacggt 1accccctagtagtcacctgtgctggtgaagaaccacctagtattgcagggtgcgtgccgcctagcttccatttcttctggagcactgtgcaatgaggtaccccacaggtaacaagtgcctcaggtcccgcaaggatactgtggggtggtgtgaccgcagggagctgtctccacggctcctctaatgttacgccgctatccacaggccagtgcgtgtcatcgatcccggatgacagagctagtattgcgaacccccaagtaagaaaagtggctagtaacctgatagctggtgaagagggtgggtcagttgagtagatgccctagaggtacccgaaaggatctgactagggacccgtgactatacattaggtaaaccgggtataaaaaccatgaaaaactgaccactatcattaacctcactactattatgtgtgaaagtgattgagtaggactgtaccagcccgcctttcttggattttctctatcgctcttcttacacctactgttatcaaggcactctttagagata645 Kunsagivirustttcaaatcggactccggtagttataccggagcccggtaggacgcagggccgcgttaacagccc 1cccaccccatcccactgactgatactcggattggactcatagcattgctaactctgattctggataccccgtttatgtcgtcgcggtcggaagtgcacgtacacgacgagatctgatggcgatgtaccaggggggaggtggcggcagaaacgcccccgccgtaaacacggcgggccacgcctgtcaagccactccctggggccgagcgcctgaggtgatacagagagataagcacactgggcgctgacaacgcccgggacctcagtgagaagagcagtagggccgtgatatgggactccattggatatcccccgcagtcggaactcacggctactccgggagggaagcccgcgactggtactgtactgggtgatagcctggtgccttccctctcactgagtatgaaggctgaaaaccccct 646 Kagoshima-tatagtagcctgatctcgcaccgttaccgctcgacgggaatgtgaaactggcacccctcctctccc2-24-KoVctaccaccctactccacgcccccattcataatttacaacgccgcacacagcggcggccgccaagggctagcctggcggaataaaaggaacctgggtctaccctcacaagccaaaaggtaggaccctgtgtccctgaatgctcggtgaggaatgctgcaccgtaacgctagtgaagtgtttgcaagttctggcccggcaagcctacagagtgctgtgatccgctgcggacgccatcctggtaacaggacccccagtgtgcgcaacagtatgacagacacggatgacacagcatcatggaccattgcgcgaaagtgcgtgcgccatatccctgtacacaggtgtgcactctggaccctaggaatgctgcgaaggtaccccgatcggcgggatctgatcgcaggctaattgtctatgggacagtaccatacatactccacaattgactgcttaactgactctggatcagtgcaccactgctctactgctctcaaaacggcacacttaccaactctcaccatcgaccaacaccatttacacactaactatacgactcactgactcctggcaggtgaagac 647Kashmirbeetacgtacaattagacgcacgacatgcaacaatgaactcacatgtggcgctcggtagtaaccagag virusgggcgtcattcccccgtatggagtggagaatataagctaccgactcgagctgtagaataattcagcaacttataacgaacacgaatatagtcgacgaaaccatatagccataaactatgatagattataatgataaacagctcatgtaactgtctaaactacataaatacaactggattacgaaccataagtaactatcaagatgaagtctagtagtctcccaatataccgtgaaaagaatgagggacgagatagctctatttaaagacgtgaggcataaaactgataaatacattacctgagaaacctccataggagaagaatagaaaagattagtacctatacttaatagaaataaatatgaaataatgctgaagacaagtatacgattattaacctctatatactatataaagtatctgtgagtctcagtggacatcacagtaaggtcgcagaaacagtaatcttttcattcctgtgtcggagcagtggtaatggagccggacgatacgccaaaac 648 Jingmentgtgtttttgtcaagataattgttctgtgattaacagtgattgtggttcgtgtaatgcgacgcagtcaaatpicorna-gctagttttgatgaagtgtatgagagagtggaaaacttatctcataagaagattgaagagtgtgtagalike virustcaggctattgatcgagcttctaagcttcgtgattacaagcttaatgttcacaatggctcccgacgggaatcatctgatcctctctttatttcgccccattcgttgttatcgcttggggtatctaagtttgttgcgtttgagcagcatcacagtttcgcttcagttgagtctctgaagttgcttgctctgtct 649 Mumpsaccaaggggaaaatgaagatgggatgaggtagaacaaatagtgtaagaaacagtaagcccgga virusagtggtgttttgcgatacgaggccgggctcgatcctcaccatcattgtcgataggggacattttgacactacctggaaa 650 MousegaagttgatcatgaacttggttattggtggaacgcacatgaactcccaacaatgatcttgaagacacMosavirusagcgtggtaacaattaccatgcccagtggctgcccaagacattgatggctattgggtgatttatgat gac651 Miniopterusgttgtcgacccgttgatggataagcatgaggtggattcccccgattatgtctacccgttactatggcschreibersiigggcggtcgatcagggtgatctactgaggactgcaccaagtattatcattattctcagatctccggpicornavirusctgtttgacgcagtaggacagcaggactattactcttaatctattctacccactagggtcctatccta 1gtggagggagggtgccacccatctctattagagagtgcgcctggcggtctaccgtctctggaaaaaggagcacatggcatgctacaattggcacaagaaaacaagctagcggattattctagtactagaggaagctgtagcgaccctgtatggcgagcggactcccctctcggcgacgagagcctctcgggccaaaagccaagtgttaatagcacccatacaggcggcagtaccccactgccctactcaacatacaatgactgatgaaccactgaggatactgacacctagtagtaggattccaaggaatgtcctgaaggtaccctgttagcttacgcgcaggatctgatcaggagtctattacagtgctgtacactgtgcaagggattaaaaattgtttgaggaatccccgagatagtggtctatctatcctattagattacagacacg 652 Lindagtatagcagcagtagctcaaggctgctatacgattggacataccaaattccaattggtgttagggacvirus cacctaggtgaaggccgacgacaggtagccattcctgttagtaggacgaaccgttatggtggactggttgctcaggtgagcaggctgcaatgcgtaagtggtgagtacaccacagccgtcaaaggtgccactggtaaggatcacccactggcgatgccttgtggacgggggcgtgcccaacgcaatgttagcggtggcgggggctgccatcgtgaaagctaggtcttgatggaccttgttgcctgtacagtctgataggatgccggcggatgccctgtgacagccagtataaagaatatccgagtgattgcac 653 Lesavirustctttctttattttcttatgtaactcactattaagattattagcctacttgtgagcttatgcgggaccactg2 tcttagacaaccccacatagtcatgagtaagtacacgcaaccattacgattactattaaccgtctgaccttttgataacaactgaagttaggcgtgaaacatgcatttataccaaagtagccccgcatttccccactacggtgggggggctaccctactggctaggaactgtagccattatgtgagcctggctttcaggatctcacaacacaacagactctcacaatggaatatgggtgagattgcagtgacatgaacaagtatctagtagtacatagactcaagcctagttgcctgcggaacaacatgtggtaacacatgccccagggtccaaaagacaagggttaacagcccatctaggtgtctgtgtgtgaagaatactttagtagtgttgttatgatctcacctgttagtacagaatgagtatggcaggtgaaggatgtcctacaggtacccattatatggatctgagtaggagaccactagtggtggctttaccgccaggtgagtggtttaaaaagcgtctagccaagccaacagcactagggatagtgctactatatatatattacagtgtatatggtgacaa 654 Lesavirusgtaactaataagcaagattactgcctgcaaactgcttcaatgggaccaccgcttcggcgacccatt 1gttgagtttgtatgtttttaagtaatattgcaaccatacgattatatagccgcctctctataatgatcagttatagtgggacgtgaaacattggatactcacacacgtccggtcacccgggcgtgtgacttccgtaagtcctatccacataccatcgtgggtaggccagcatgatgcacaggctgtgacagtgtgggtgggctttccacctctcaacaacacactgaattgcaatgcactcacggaggaaatgacaataggttatagttttgaactgtgctagtaatttattcacattaagccatgagcctgcggaatcacatgtggtaacacatgcctcagggcccaaaaggcacgggttagcagccccttcatggtgtgttagaagtgaaaacacatagtatgagctataatatattgagtatctctgtagtgtaccccgccaaatgtaaggatgcccagcaggtacccatatatggatctgagctggggattgatagtgtatctataaatgcactgatcaatttaaaaagcgtctaagtaggcacaaacactggggacagtgatttcattattatttatttgatta 655 PhopivirusgggagtaaacctcaccaccgtagccgtggatacggctacctatattggatgtaaatattaattcctgstraincaggttcaggtctcttgaattatgtccacgctagtggcactctcttacccataagtgacgccttagcgNewEnglandgaacctactacacttgatgtggttaggggttacattataccctgggccactaggccattacccctgcactatcattattcaccgggctctcagcatgccaatgaccgaccggtgcgcccgccggggttaactccatggttagcatggagctgtaggccctaaaagtgctgacactggaactggactattgaagcatacactgttaactgaaacatgtaactccaatcgatcactacaaggggtaggctacgggtgaaaccccttaggttaatactcatattgagagatacactgataggttaaggagctggataatggtgagtttaacgacaaaaaccattcaacagctgtgggccaacctcatcaggtagatgcttaggagccaagtgcgtaggggtgtgtgtggaaatgcttcagtggaaggtgccctcccgaaaggtcgtaggggtaatcaggggcagttaggtttccacaattacaatttgaa 656 Pestivirusgtatacgagattagctcatactcgtgtacaaattggacgtagcaaatttaaaaattcggatagggtccstrain ccatccagcgacggccgaacggggttaaccatacctctagtaggactagcagacggatggactaAydin gccacagtggtgagctccctgggaggtctaagttctgagtacagaacagtcgtcagtagttcaacgctggtaaaccccagccttgagatgctacgtggacgagggcatgcccaagacacaccttaacctggacgggggtcgtccaggtgaaagtacccatctagggtgctgggagtacagcctgatagggcgctgcagaggcccactgacaggctagtataaaaatctctgctgtacatggaac 657 Quailtttgcatcagttcgcccctcccctcaccataccatttcccctattaggactgatacttggttatgatgapicornavirusgcagaggatttcgcaagttatgatcttgataaaaagtaattcacgaatcatgggattatagcctgga QPV1agtgaacactcatgtggcaagtgggttagtagctctccatgacccatgtgcagtggactgacaacagtgagttcggggagtgtagtaaagggaaagtattacttacccgcacctgctatacgtggtgtacgtaggatacgagttagtagtgcttagcaactttaaactggtgctgaaatattgcaaggtcactgaagttgtgaacgcgaacgctccgccactgccatgtatagcgtgcaatgcataaatggtgcactacatgatacgagggaatgggaaaccctccatggccgaatgcagggtgacagcctgccggcggatgcctgttgttagtataatccgttgatgccac 658 Porcineacactcatttcccccctccacccttaaggtggttgtatcccctacaccctaccctcccttccacataggsapelovirusacgaataaacggacttgagattaaggcaagtacataaggtatggataggatacacttaaatggca 1gtagcgtggcgagctatggaaaaatcgcaattgtcgatagccatgttagcgacgcgcttcggcgtgctcctaggtgattcggcgactggttacaggagagtagacagtgagctatgggcaaacccctacagtattacttaggggaatgtgcaattgagacttgacgagcgtctctttgagatgtggcgcatgctcttggcattaccatagtgagatccaggagggaaacctggactgggtctatactgcctgatagggtcgcggctggccgcctgtaactagtatagtcagttgaaaacccccc 659 Porcineatgacgtataggtgaggctctatgccaggcatagtattgtcaggagctgtgaccattggcacagcreproductiveccaaaacttgctgcacagaaacaccatctgtgatagcctccttcaggggagcttagggatgtccct andagcaccttgcaccggagttgcactgattacggtctctccacccattaacc respiratory syndromevirus 2 660 PorcineGaaccttagaagatacacaaacaaagaccaataggagtccaacacccagaggattgcggtcaaenterovirusgcacttctgatccccggacctagtagtgataggctgtacccacggccgaagatgaacccgtccgtt 9atccggctacctacttcgggaagcctagtaacattctgaagtctctgaggcgatcgctcagcacgaccccggtgtagatcgggctgatgggtctccgcataccccacgggcgaccgtggcggaggccgcgttggcggcccgcctatggcgaaagccataggacgcctcttagatgacagggtgtgaagagcctactgagctgggtagtagtcctccggcccctgaatgcggctaatcctaaccacggagcgtccaccagcaatccagctggcagggcgtcgtaacgggcaactctgtggcggaaccgactactagggtgtccgtgtttccattgatcctattaggctgcttatggtgacaacgataagagttatcataaagctcttgggttggccacctggaaaaagttatcagtgatgatattgacggctctcacgcctaccaataagacaagccctatatttacttgagcatatactcgtcagaagaaatcacagagtatcataggatagttactcacattaaggacaag 661 PigeontttatttagctgttaagattatttgtgccgagAccccatagtaggatcaggtgaccacattaagctctpicornavirusCccgaccacacatccaaacgataggcggtgtaagggctccctggctaagtgatactcattgctag Bggaagtgagcgacccgttaccagtaggaatacaggaggtcttagttgcctaaccagataaagtggtgctgaaatattgcaagctcaatgtctggcgaacgacggactaccgttgaactattgttaacgcccgcgtgtgtaggcaacacacgggttagtaggtcacttacattgacatccgtgccgggaaagcggatctgagctatcgattgcctgatagggtgccggcgggcgcggtacgtgtggtatagtccgctgtcttggggtatggcgtctactcggattgagtcgattggaatgtcccatgtcggagatgtcgttaccggtgtttgctttacttgtgcacgaataagaaaacagagtaggagattggaagttaatggataacatcaagtaccattgcgacctcgtgcatataggccagaaaatcgaccacaaaatcgaga 662 Picornavirusaaacgggaggatcggctaggcttatcctcttaatagtctacaaaactgggctgactggtggggga HK21gctactagatccgggagaggactagttaccccgcgtaacttccattagtcactccctccacctacccatccctgtcccttagactcttaagtaggtgtgcaggcctggccccccggaaatgggcaaatcggacgtttgcgtgtagggtcgactgaaaggtggggcccactccaccgtagtaggatcacctgatcacgtttaaacctctccgggcaggtatcgctagacactaggctgtataggatggcacgcacttaggatcgcaccacctagtgcgccaagatcccgcattgagctcaagtacatggactagtaagatgtctaaccagaggaggtggtgctgaaatattgcaagccactctggcgaacgtccactgcattgcctggaacaggctctctagcgccctccactggtagcgcggtggtgggttagtaggatacctatatggacaggggatgcgggaatacccctcactagctagtgactggttgatcgactggcggcggatccagtgatacttgcataatccgcagacttgggag 663 Picorna-agccatgaactagtgtgcgattcccacagtgaggtcgaaagccccgcactgtctactcgcatatttvirales gactccagtccaccgcagccagcggttaggactggttaaagtgcattatgtgcacggcgccacgTottori-HG1cagaactgccagaaatggtaagctgcgcccaacgccaacggtagtgtatcccgttagtcacacgtttacagctgttcgttaacagtagggttttgtcgacccggaccccttaatgcgcgcgaattcaaccgcgcctagtcggcaatggtatcatttaatcccatgcactacgggagaaatttgagaccaaagaattcctgagggccactgcttgctctaagtgcaatgcctcgggagacttctgtcaggagcctagcggctttcaaccgcgacagctaactcctgcgggatgtttggtgtccatacttactggcgtcctcacaacgctaagtggatgttgtccacaggtaggcaaacaccgagccccacattcaggagacctgtatgaacgatcctatcagcattagagttggaattgggtgtgctaacgtccgcataagtgcaccccgtggtaacgctgggaaactatccagcgcaacgtactgtcctcaatgtctagggaaggaccgccctaagcgtacaaccgggccatgtgtcgagc 664 RodentttcaaagaggggtccgggattttcctggtccccctctttgggcacccttggctcgggggtgtgaatahepatovirusccgtgctcgcgtttgccgtgcgttaacggcttcatttatgtttgtttgtctgttttattatgttGgtttgtctgtttgttatgttggtattgttcgtgtttaatgttatgaccacattacactccagccaatgaagaacagatggtgcggttattgctggcggaattcctaacgtcctggatccgttggtacgcatcacaaaacaatttgcagagagagtggtgaaacggcttgggaatccctgagtacagggaaatcacactgatagctcatcttggctgttttcagtcatggaccttatgcagtgtaatttgggtgtaccccccatagcttaggaggaatgttctgtcttggcactagagtgggacgctgatgcctccgtgtctaggatggtctaagggacagaatggggtgcctctgatgccatactacctgatagggtgctctcacggcctctgcatcttagtgagaagttcaattt t665 Rinderpestaccaaacaaagttgggtaaggatcggtctatcaatgattatgatttagcacacttaggattcaagatcvirus ctatcgactggagcaggcttaaggtaaaggttctttaaa 666 Rabovirusctacggatatttgcatgacccgctttctatcgccccaacaatcccctttgtaaccacaagctttactca Aggctagcagcccgactagctgtttggaagaaaaggctagggcacacaccaacaacaccgaccccactggtcgaaggccgcttggcaataagactggtggaacagggtcgcctgtagttgtttggaacattctttctaatgactttgtcagcggtgctactcacaccgtaactcttctaccctatccccacgcttgtggaactaggaggggatgagtgattcaagtaagtactgtcagaatggtgaaaatgatctgattctgaaacgctatggatccatcgaaagatggggctacacgcctgcggaacaacacatggtaacatgtgccccaggggccgaaagccacggtgataggatcacccgtgtagtttgagatcatatcaatgttcatagtctagtaagatgatttgaaatctaactgagctgatggctaactgcttgtcttattgcggcctaaggatgtcctgcaggtaccttagataaccttaagagactattgatctgagcaggagccaaagtggtctttcccagctttggttaaaaaacgtctaagccgcggcagggggcgggaggccccctttcctcccaaaacttaatatt gattgt667 Shinglebackctgtgagtaccgacaggctcgaagtctattatgaggcgtcgaaacagaaaacctgtaacaactccgnidovirus 1gtttcatctatcactgccgtcaagaggcagaagaggacgaccacgtgtcaccagatcacttgtatctgtttcagtcaggaagtcaacttttcgacgaagttcgaccattcatcgacccgctgaaaagcgtagaatcgagctcgtgaatttcgagctgaaacaacaaatcaacgagaacgagtggtacggttattataattacgacaaccaaaactgcaaagttcagttgccagtcacatgtcgaatcgaggacgtaacctgggatcaggtttacgtg 668 Senecatttgaaatggggggctdgggccctgatgcccagtccttcctttccccttccggggggttaaccggctgvalleytgtttgctagaggcacagaggggcaacatccaacctgcttttgcggggaacggtgcggctccgatt viruscctgcgtcgccaaaggtgttagcgcacccaaacggcgcacctaccaatgttattggtgtggtctgcgagttctagcctactcgtttctcccccgaccattcactcacccacgaaaagtgtgttgtaaccataagatttaacccccgcacgggatgtgcgataaccgtaagactggctcaagcgcggaaagcgctgtaaccacatgctgttagtccctttatggctgcaagatggctacccacctcggatcactgaactggagctcgaccctccttagtaagggaaccgagaggccttcgtgcaacaagctccgacacagagtccacgtgactgctaccaccatgagtacatggttctcccctctcgacccaggacttctttttgaatatccacggctcgatccagagggtggggcatgacccctagcatagcgagctacagcgggaactgtagctaggccttagcgtgccttggatactgcctgatagggcgacggcctagtcgtgtcggttctataggtagcacatac aaat669 SclerotiniattgaattaatcttttacgtttacgcgcataaaatcaggacacatctcttgtatactttagtatatcaatgatsclerotiorumgtttttgttttatgcgattaatcgtaagagaacttctttccatccgcctgtatgggcgggataataagttcdsRNAaccgccttggtcgaggcgcaaacttgtatgtgcaaaggtgagctatatgctcgaaatagtcgtaactmycovirus-Laacacacagccactacctgtagagctctattgatccggaatcctttagtgggaatgcagagctcaccggacctgcgggttcttcggcgttagggacttctgtttcagccttgaatcatttacctttataccttctctgaggcgcctgggccgggcgcgatattaagtacaagtcaaggacatcgcgggtagtggtctaatcagccgctagtcctgctggagagttccaacttagttgggtgtggtgcatactagctggatagagtaggtatgtattgctaacgtatgccggaggctatccgtcctcggtagaacgtgccgaggagtagtctctgcagacccccgaacgcgtggggtctttacttaaatgtaggcggagggagcgctcgtaggtggaacgactgcctcccagtcgaatgcaagattttgcacgcggaccagtctgcccggcaattcccgggtg 670 Yakctccggcacagccgcaccagtgcactggtacgctagtaccttttcacggggtagtcggtatcccccenteroviruscccgtaacttagaagcatgtaacaaaccgaccaataggtgcgcggcagccagctgcgttgcggtcaagcacttctgtctccccggtccgcaaggatcgttacccgcccactccactacgaggagcctagtaactggccaagtgattgcggagttgcgttcagccacaaccccagtggtagctctggaagatggggctcgcacatcccccgtggtaacacggttgcttgcccgcgtgtgcttccgggttcagtctccgactgttcacttcaacatcacgcaaccagccaagagccgattgtgctggagtggtcttcctccggggccgtgaatgctgctaatcctaacctccgagcgtgtgcgcacaatccagtgttgctacgtcgtaacgcgtaagttggaggcggaacagactactttcggcaccccgtgtttcctttattttattcttattttatggtgacaattgcagagatttgtgatattgcgactttaccgttaaacatagcactgcattacctggttgcattccacaaaacttcagagattcctagttcctacattgacctacttgtttatttgaatcttaaatacaaacttgagcaagtgaa 671 Wobblycggctgtgagtgcttagcatatgctagagtactacagccgggtgttggagtcatatgcactggttgcpossumctgtataatagtcgggatctgtctgacctacattatctttgggagttgcttatcacgacaattctcgaagdiseasetgtctgtcgacagcttacccccgattcgacaaggccccttgtccaccgcagacctatcgattttcaacvirus gagaacactatcagaggtttaaatttaaaactcaccaaca 672 AviangctttttcaatcccttgtgtcgatgttcccgtatgtcatctggttcatgtaacggtgcaacttctattttgorthoreovirusgtaacgttcactgtcaggcagcgcaaaactcggcgggtggtgatcttcaagcgacttcctctcttgttsegmentgcttattggccctacttggctgcgggcggtggtctgctcgttgttattattataattgttggcgctgtttgS1 ttgttgcaaggctaaggttaaagcggacgcagcccgaaacgttttctaccgagagctgttcgcacttaattcgggtaaaagtgatgcaggacctccgatttaccaggtttagtgtacgacgatttgagttttcacctttcgtcttagaggagtgcactactccatctttcacgactataactaataccgatccggctctctactttaacattgagtttccgtcaagtcatcgtctctcccccttcattccagaactgttgtctcagccttgtaccgttcacgtttcattgattcggagattcgctctctgtgcaaccttatctagtatttgtgaatacgactgtgcgctactgccatccatcaacgctattacgacgatccctacaccaggtgcgtcatcatctctgattgttcattggg 673 CaprineggggcctcggccccctcaccctcttttccggtggccacgcccgggccaccgatacttcccttcactKobuvirusccttcgggactgttggggaggaacacaacagggctcccctgttttcccattccttcccccttttccca d10accccaaccgccgtatctggtggcggcaagacacacgggtctttccctctaaagcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcgggagtgctcccacccaactgttgtaagcctgtccaacgcgtcgtcctggcaagactatgacgtcgcatgttccgctgcggatgccgaccgggtaaccggttccccagtgtgtgtagtgcgatcttccaggtcctcctggttggcgttgtccagaaactgcttcaggtaagtggggtgtgcccaatccctacaaaggttgattctttcaccaccttaggaatgctccggaggtaccccagcaacagctgggatctgaccggaggctaattgtctacgggtggtgtttcctttttcttttcacacaactctactgctgacaactcactgactatccacttgctctcttgtgcctttctgctctggttcaagttccttgattgtttttgactgcttttcactgcttttcttctcacaatccttgctcagttcaaagtc 674 CaprineccccctcaccctcttttccggtggccacgcccgggccaccgatacttcccttcctccttcgggactKobuvirusgttggggaggaacacaacagggctcccctgttttcccattccttcccccttttcccaaccccaaccg d20ccgtatctggtggcggcaagacacacgggtctttccctctaaagcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcgggagtgctcccacccaactgttgtaagcctgtccaacgcgtcgtcctggcaagactatgacgtcgcatgttccgctgcggatgccgaccgggtaaccggttccccagtgtgtgtagtgcgatcttccaggtcctcctggttggcgttgtccagaaactgcttcaggtaagtggggtgtgcccaatccctacaaaggttgattctttcaccaccttaggaatgctccggaggtaccccagcaacagctgggatctgaccggaggctaattgtctacgggtggtgtttcctttttcttttcacacaactctactgctgacaactcactgactatccacttgctctcttgtgcctttctgctctggttcaagttccttgattgtttttgactgcttttcactgcttttcttctcacaatccttgctcagttcaaagtc 675 CaprinectcttttccggtggccacgcccgggccaccgatacttcccttcactccttcgggactgttggggaggKobuvirusaacacaacagggctcccctgttttcccattccttcccccttttcccaaccccaaccgccgtatctggt d30ggcggcaagacacacgggtctttccctctaaagcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcgggagtgctcccacccaactgttgtaagcctgtccaacgcgtcgtcctggcaagactatgacgtcgcatgttccgctgcggatgccgaccgggtaaccggttccccagtgtgtgtagtgcgatcttccaggtcctcctggttggcgttgtccagaaactgcttcaggtaagtggggtgtgcccaatccctacaaaggttgattctttcaccaccttaggaatgctccggaggtaccccagcaacagctgggatctgaccggaggctaattgtctacgggtggtgtttcctttttcttttcacacaactctactgctgacaactcactgactatccacttgctctcttgtgcctttctgctctggttcaagttccttgattgtttttgactgcttttcactgcttttcttctcacaatccttgctcagttcaaagtc 676 CaprinegtggccacgcccgggccaccgatacttcccttcactccttcgggactgttggggaggaacacaacKobuvirusagggctcccctgttttcccattccttcccccttttcccaaccccaaccgccgtatctggtggcggcaa d40gacacacgggtctttccctctaaagcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcgggagtgctcccacccaactgttgtaagcctgtccaacgcgtcgtcctggcaagactatgacgtcgcatgttccgctgcggatgccgaccgggtaaccggttccccagtgtgtgtagtgcgatcttccaggtcctcctggttggcgttgtccagaaactgcttcaggtaagtggggtgtgcccaatccctacaaaggttgattctttcaccaccttaggaatgctccggaggtaccccagcaacagctgggatctgaccggaggctaattgtctacgggtggtgtttcctttttcttttcacacaactctactgctgacaactcactgactatccacttgctctcttgtgcctttctgctctggttcaagttccttgattgtttttgactgcttttcactgcttttcttctcacaatccttgctcagttcaaagtc 677 CaprineccgggccaccgatacttcccttcactccttcgggactgttggggaggaacacaacagggctccccKobuvirustgttttcccattccttcccccttttcccaaccccaaccgccgtatctggtggcggcaagacacacgg d50gtctttccctctaaagcacaattgtgtgtgtgtcccaggtcctcctgcgtacggtgcgggagtgctcccacccaactgttgtaagcctgtccaacgcgtcgtcctggcaagactatgacgtcgcatgttccgctgcggatgccgaccgggtaaccggttccccagtgtgtgtagtgcgatcttccaggtcctcctggttggcgttgtccagaaactgcttcaggtaagtggggtgtgcccaatccctacaaaggttgattctttcaccaccttaggaatgctccggaggtaccccagcaacagctgggatctgaccggaggctaattgtctacgggtggtgtttcctttttcttttcacacaactctactgctgacaactcactgactatccacttgctctcttgtgcctttctgctctggttcaagttccttgattgtttttgactgcttttcactgcttttcttctcacaatccttgctcagttcaaagtc 678 Picorna-tttgctcagcgtaacttctccgggttacgtggagaccaaaaggctacggagactcgggctacggcviralescctggagcacctaggtgctcctaaagacgttagaagttgtacaaactcgcccaatagggcccccc sp.aaccaggggggtagcgggcaagcacttctgtttccccggtatgatctcataggctgtacccacggisolatectgaaagagagattatcgttacccgcctcactacttcgagaagcccagtaatggttcatgaagttgatRtMruf-ctcgttgacccggtgtttcccccacaccagaaacctgtgatgggggtggtcatcccggtcatggcg PicoVacatgacggacctccccgcgccggcacagggcctcttcggaggacgagtgacatggattcaaccgtgaagagcctattgagctagtgttgattcctccgcccccgtgaatgcggctaatcccaactccggagcaggcgggcccaaaccagggtctggcctgtcgtaacgcgaaagtctggagcggaaccgactactttcgggaaggcgtgtttccttttgttccttttatcaagttttatggtgacaactcctggtagacgttttattgcgtttattgagagatttccaacaattgaacagactagaaccacttgttttatcaaaccctcacagaataagataaca 679 ApodemusttactcagcgtaactactccgggttacgtgatgaagaagaggctacggagattctcgggctacggcagrariuscctggagccactccggctcctaaagatttagaagtttgagcacacccgcccactagggccccccapicornavirustccaggggggcaacgggcaagcacttctgtttccccggtatgatctgataggctgtaaccacggctstraingaaacagagattatcgttatccgcttcactacttcgagaagcctagtaatgatgggtgaaattgaatcLongquan-cgttgatccggtgtctcccccacaccagaaactcatgatgagggttgccatcccggctacggcga Aa118cgtagcgggcatccctgcgctggcatgaggcctcttaggaggacggatgatatggatcttgtcgtgaagagcctattgagctagtgtcgactcctccgcccccgtgaatgcggctaatcctaaccccggagcaggtgggtccaatccagggcctggcctgtcgtaatgcgtaagtctgggacggaaccgactactttcgggaaggcgtgtttccatttgttcattatttgtgtgtttatggtgacaactctgggtaaacgttctattgcgtttattgagagattcccaacaattgaacaaacgagaactacctgttttattaaatttacacagagaagaattaca 680 Niviventerccctttcataaccccccccttttaacccaacccttcgtaaccgtacgcttcactcgcctttgggtatagconfucianuscggcccaatgtgctgaagaaaaggatacgctataaggggccaacgggtggtggcccttaagaccpicornavirusacccaacctagaagcttgtacactcgggcaatagtgaggcccacatccagtgggtcaagcccaaagcattcttgaccccggtatgatctcataagctgtacccacggctgaaagagtgattatcgttatcccactcagtacttcggagagcctagtacaccacttggaaatggaagtctgtgatccggggttgaccctgaaccccagaaactcatgatgaggctaaccttcccgaacacggcgacgtgtggttagcctgcgctggcatgaggcctctttgtaggcagactgaaatggaagggtgacgaagagccgactgagctactgttttattcctccggccccctgaatgcggctaatcctaactcctggtccagtacttgtaacccaacaggtggctggtcgtaatgcgtaagccgggagcggaaccgactactttggggcgtccgtgtttctcaatattattcatttctagcttatggtgacaatttatgattgcagagattgtgctgtatttgtgtctgagagaagaagtaacaat 681 BattttcaaaaggccctgggcatacggcgttattcgtaacgtcgtatgtccagggcggtagcatcaggcpicornaviruscaaggcctgatgctaccacgtgtggactaaaccacacactcttcttgtgacacgttgtgtcacctatcisolatecctttcttggtaacttagaagcttgtacacttacgcacgtaggtgccccacatccagtggggtttgtgBtRs-caaagcaatcttgttccccggtaaaccctgataggctgtaaccacggccgaaacaaggtttgtcgttPicoV acccgactcactactacacaaagcctagtaaagacaatgaaagtgcgcagcgtgatccggtcaaaacccccttgaccagaaacacatgatgagggtcaccaacccccactggcgacagtgtggtgtccctgcgttggcatgtggcctcgtagaggcgagcaatctggatttgctccgaagagccccgtgtgctagtgtttatacctccggccccttgaatgcggctaatcctaacccccgagcatgtacacacaagccagtgtgtagcatgtcgtaatgagcaatttggggatggaaccgactactttagggtgtccgtgtttctcattattctttgtttgatgtttt 682 RhinolophusttttttttctcaggcggtagcatccagccaaggcctgatgctaccaacgtgtgactaaaccacactctpicornavirusctttttgtgatacattgtgtcacctatccctttcttggtaacttagaagcttgtacacccacgcacgtagstraingtaccccacatccagtggggttgtgcaaagcattcttgttccccggtaaaccctgataggctgtaaGuizhou-ccacggctgaaacaaggtttgtcgttacccgactcactactacgcaaagcctagtaaagttcaatgaRr100 aagtgcgcagcgtgatccggtcaaaacccccttgaccagaaacacatgatgagggtcaccaacccccactggcgacagtgtggtgtccctgcgttggcatgtggcctcatagaggcgttgcaatctggatttgctccgaagagccccgtgtgctagtgtttatacctccggccccttgaatgcggctaatcctaacccccgagcatgtacacacacgccagtgtgcagcatgtcgtaatgagcaatttggggatggaaccgactacatttgggtgtccgtgtttctcattattctttgtttgatgttttatggtgacaaca 683RhinolophuscggaacgttgtatgctcagggcgtaggcaccacccacgggtggtgcctacacgtgtggactaaacpicornaviruscacacactcttttcagcacttagtgctgctatctctttttgtaacttagaagtttgtacacaatgcgttagstrainggccacacatccagtgtggtatcgcaaagcacttctgtttccccggtgctagtaggagggtggctgHenan-ctccacggccacttgccgaacccatcgttacccgactcattacttcgcaaagcctagtaacccagttRf265 gaagcaagcccggcgtgttccggtcaggaaaaaccccccctggccagaaacatgtgatgagggtgggctatccccactggtgacagtgagccctccctgcgttggcacatggcccgatctgggcgtggttcttgtggatgctgccgaagagccccgtgagctagtgtttataccgccggcctcgtgaatgcggctaaccctaaccccggagcagaggctactgaagccacagtagtcgctgtcgtaacgagtaattctgggatgggaccgactactttcgagtgtccgtgtttcctttattcttttattgttgtttatggtgacaaac 684Humancccctaggatccactggatgtcagtacactggtatcgtggtacctttgtacgcctgttttatacccccttenterovirusccccgcaactttagaagcatcaaaagcaccgctcaatagtcaccacacccccagtgtggtttcgag C105caagcacttctgttttcccggttgcgtcccatatgctgtgcaaacggcaaaaagggacaatatcgttacccgcttgtatactacgggaaacctagtaccaccattgattgtgttgagagttgcgctcatcacctttccccggtgtagctcaggccgatgaggctcagaatcccccacaggtgactgtgtctgagcctgcgttggcggcctgccctcgccttatggcgtgggacgcttgatacatgacatggtgcgaagagtctactgtgctatgcaagagtcctccggcccctgaatgtggctaatcctaaccactgatcccacgcacgcaaaccagtgtgtagtgggtcgtaacgcgcaagtcggtggcggaaccaactactttgggtgaccgtgtttcctttattacttattgaatgtttatggtgacaattgtttgattcagttgttgccattctctacattcatttacccagcatcaaaccaattgaactgttaca 685 Humanagtctggacatccctcaccggcgacggtggtccaggctgcgctggcggcctacctgtggtccaaapoliovirus 1gccacgggacgctacatgtgaacaaggtgtgaagagcctattgagctacaaaagagtcctccgg straincccctgaatgcggctaatcccaaccacggatcaagggtgcacaaaccagtgtacaccttgtcgtaNIE1116623acgcgcaagtctgtggcggaaccgactactttgggtgtccgtgtttcctttttaattttgatggctgcttatggtgacaatcatagattgttatcataaagctaattggattggccatccggtgagagtgaaatatattgtttacctccctgttgggtttactctaactaacttctccatttataaacttgtcatcacagttttaataattagaagtgcagtttaca 686 Humantttaaaacagcctgggggttgttcccacccccagggcccactgggcgttagtactctggtatcgcgenterovirusgtaccttagtatgcctgttttatgtctcctttcccccgcaactttagaagtaatcaagttatggctcaaca109 gtcgccacacccccagtgtggttccgagcaagcacttctgttccccggttgcgtcttatatgctgtgtgaacggcagaaagggacaatatcgttatccgctcaactactacgggaagcctagtaccaccatggattgacctgaaagttgcgttcagcgcacccccagcgcagctcaggccgatgaggctccgaataccccacgggcgaccgtgtcggagcctgcgttggcggcctgcccacgttgcaaaacgtgggacgctcatttcatgacatggtgcgaagagcctactgtgctagttgagagtcctccggcccctgaatgtggataatcctaaccactgaacctacgggcgcaaaccagcgtctggtaggccgtaacgcgcaagtcggtggcggaaccaactactttgggtgtccgtgtttccttttatctttttgaatgtttatggtgacaattgttgtgtacagttgttaccatagtttgcattcagaaataaacctaacactttccaattatttgttaca 687Humanttgtgcgcctgttttatattccccccccgcaacttagaagcacgaaaccaagttcaatagaagggggpoliovirus 2tacaaaccagtaccaccacgaacaagcacttctgtttccccggtgacattgcatagactgctcacgcstrainggttgaaagtgatcgatccgttacccgcttgtgtacttcgaaaagcctagtattgccttggaatcttcgNIE0811460acgcgttgcgctcagcacccgaccccggggtgtagcttaggctgatgagtctggacattcctcaccggtgacggtggtccaggctgcgttggcggcctacctatggctaatgccataggacgctagatgtgaacaaggtgtgaagagcctattgagctacataagagtcctccggcccctgaatgcggctaatcctaaccacggagcaggcggtcgcaaaccagtgactagcttgtcgtaacgcgcaagtctgtggcggaaccgactactttgggtgtccgtgtttcctgttatttttattatggctgcttatggtgacaatcagagattgttatcataaagcgaaaggattggccatccggtgagtgttgtgtcaggtatacaactgtttgttggaaccactgtgttagtttaacctctctttcaaccaattagtcaaaaacaatacgaagatagaacaacaatacta ca688 Bovinettttctcccctccccctccaactaccttttccccctcttgtaacgctagaagtttgtgcaaaccgcctgtpicornavirusagggtactgcaatccagcagtgcataggctaagcttttcagaaccccaccccacattatactgaggaggattgtgaaattgtgttagtatgggttagtagcggtgacccgggtaaccccaacccagaaactcacggatgagatgaacaggaccccacatggtaacgtgtgtgttcgtctgccccgcaaggtgaggccgtgagagctttgcacgcgaaaaccttgaaaacccaaaagtaccttgagctcttcgctattttgtgtttcctccaggaccctgaatgcggctaaacctaacccgcgatccgcacgtagcaacccagctagagtgtggtcgtaatgcgcaagttgcgggcggtaccgactactttggtgttcctgtgtttcctttattttattttgaatttttatggtgacaacagctagaaaataagagtgaac 689 Humanacccttgtacgcctgttttatactcccctccccgtaacttagaagaaacaaaataagttcaataggagpoliovirus 1ggggtacaaaccagtaccaccacgaacaagcacttctgtctccccggtgacattgcatagactgtcstraincccacggttgaaagcaattgatccgttacccgctcttgtacttcgagaagcctagtaccatcttggaaEQG1419328tcatcgatgcgttgcgctccacactcagtcccagagtgtagcttaggctgatgagtctggacattcctcaccggcgacggtggtccaggctgcgttggcggcctacctgtggcccaaagccacaggacgctagatgtgaacaaggtgtgaagagcctattgagctataagagagtcctccggcccctgaatgcggctaatcccaaccacggatcaagggtgcacgaaccagtgtataccttgtcgtaacgcgcaagtccgtggcggaaccgactactttgggtgaccgtgtttccttttattatttcaatggctgcttatggtgacaatcattgattgttatcataaagcgaattggactggccatccggtgaaagtgaaacatattgtttgcctcctcgttgggtctacttcaaccaatctttacttacaatcttaccactacagttttgctggttagaagtgtgtttcacg690 Humanttgtgcgcctgttttatactcccctcccgcaacttagaagcacgaaaccaagttcaatagaagggggpoliovirus 2tacaaaccagtaccactacgaacaagcacttctgtttccccggtgacattgcatagactgctcacgcisolateggttgaaagtgatcgatccgttacccgcttgtgtacttcgaaaagcctagtatcgccttggaatcttcIS_061 gacgcgttgcgctcagcacccgaccccggggtgtagcttaggccgatgagtctggacattcctcaccggtgacggtggtccaggctgcgttggcggcctacctatggctaacgccataggacgttagatgtgaacaaggtgtgaagagcctattgagctacataagagtcctccggcccctgaatgcggctaatcctaaccacggagcaggcggtcgcgaaccagtgactggcttgtcgtaacgcgcaagtctgtggcggaaccgactactttgggtgtccgtgtttcctgttatttttatcatggctgcttatggtgacaatcagagattgttatcataaagcgaattggattggccatccggtgagtgttgtgtcaggtatacaactgtttgttggaaccactgtgttagctttgcttctcatttaaccaattaatcaaaaacaatacgaggataaaacaacaatactaca 691 Coxsackie-cctttgtgcgcctgttttatgcccccttcccccaattgaaacttagaagttacacacaccgatcaacagvirus cgggcgtggcataccagccgcgtcttgatcaagcactcctgtttccccggaccgagtatcaatagaB5 ctgctcacgcggttgaaggagaaaacgttcgttacccggctaactacttcgagaaacctagtagcatcatgaaagttgcgaagcgtttcgctcagcacatccccagtgtagatcaggtcgatgagtcaccgcattccccacgggcgaccgtggcggtggctgcgttggcggcctgcctacggggcaacccgtaggacgcttcaatacagacatggtgcgaagagtcgattgagctagttagtagtcctccggcccctgaatccggctaatcctaactgcggagcacataccctcaacccagggggcattgtgtcgtaacgggtaactctgcagcggaaccgactactttgggtgtccgtgtttccttttattcttataatggctgcttatggtgacaattgaaagattgttaccatatagctattggattggccatccggtgtctaacagagctattatatacctctttgttggatttgtaccacttgatctaaaggaagtcaagacactacaattcatcatacaattgaacacagcaaa 692 Coxsackie-tttgtgcgcctgttttacaacccttccccaacttgtaacgtagaagtaatacacactactgatcaatagvirus caggcatggcgcgccagtcatgtctcgatcaagcacttctgttcccccggactgagtatcaatagaA10 ctgctcacgcggagaaggagaaaacgttcgttacccggctaactacttcgagaaacctagtagcaccatagaagctgcagagtgtttcgctcagcacttcccccgtgtagatcaggctgatgagtcactgcaatccccacgggtgaccgtggcagtggctgcgttggcggcctgcctatggggcaacccataggacgctctaatgtggacatggtgcgaagagtctattgagctagttagtagtcctccggcccctgaatgcggctaatcctaactgcggagcacatgccttcaacccagaaggtagtgtgtcgtaacgggcaactctgcagcggaaccgactactttgggtgtccgtgtttctttttattcctatattggctgcttatggtgacaatcacggaattgttgccatatagctattggattggccatccggtgtctaatagagctattgtgtacctatttgttggatttactccgctatcacataaatctctgaacactttgtgctttatattgaacttaaacacccgaaa

In some embodiments, an IRES of the invention is an IRES having asequence as listed in Table 17 (SEQ ID NOs: 1-72 and 348-389). In someembodiments, an IRES is a Salivirus IRES. In some embodiments, an IRESis a Salivirus SZ1 IRES. In some embodiments, an IRES is a AP1.0 (SEQ IDNO:348). In some embodiments, an IRES is a CK1.0 (SEQ ID NO:349). Insome embodiments, an IRES is a PV1.0 (SEQ ID NO:350). In someembodiments, an IRES is a SV1.0 (SEQ ID NO:351).

TABLE 18 Anabaena permutation site 5′ intron fragment sequences.Permutation SEQ ID NO site Sequence  73 L2-1GAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGT  74 L2-2AAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTA GCGAAAGCTGCAAGAGAATGAAAATCCGT 75 L2-3 AGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAG CGAAAGCTGCAAGAGAATGAAAATCCGT 76 L5-1 GTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAG CTGCAAGAGAATGAAAATCCGT  77L5-2 TTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGC TGCAAGAGAATGAAAATCCGT  78L5-3 TATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCT GCAAGAGAATGAAAATCCGT  79 L5-4ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTG CAAGAGAATGAAAATCCGT  80 L5-5TAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGC AAGAGAATGAAAATCCGT  81 L6-1ACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGA GAATGAAAATCCGT  82 L6-2CAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGA ATGAAAATCCGT  83 L6-3AATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAA TGAAAATCCGT  84 L6-4ATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAAT GAAAATCCGT  85 L6-5TAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATG AAAATCCGT  86 L6-6AGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGA AAATCCGT  87 L6-7GATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGA AAATCCGT  88 L6-8ATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAA AATCCGT  89 L6-9TGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAA ATCCGT  90 L8-1CAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGT  91 L8-2AAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGT  92 L8-3AGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGT  93 L8-4GACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGT  94 L8-5ACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGT  95 L9a-1AATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGT  96 L9a-2ATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGT  97 L9a-3TAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGT  98 L9a-4AGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGT  99 L9a-5GGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGT 100 L9-1GAAAGCTGCAAGAGAATGAAAATCCGT 101 L9-2 AAAGCTGCAAGAGAATGAAAATCCGT 102 L9-3AAGCTGCAAGAGAATGAAAATCCGT 103 L9-4 AGCTGCAAGAGAATGAAAATCCGT 104 L9-5GCTGCAAGAGAATGAAAATCCGT 105 L9-6 CTGCAAGAGAATGAAAATCCGT 106 L9-7AAGAGAATGAAAATCCGT 107 L9-8 AGAGAATGAAAATCCGT 108 L9-9 GAGAATGAAAATCCGT109 L9a-6 GCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGT 110 L9a-7AGTAGCGAAAGCTGCAAGAGAATGAAAATCCGT 111 L9a-8GTAGCGAAAGCTGCAAGAGAATGAAAATCCGT

In some embodiments, a 5′ intron fragment is a fragment having asequence listed in Table 18. Typically, a construct containing a 5′intron fragment listed in Table 18 will contain a corresponding 3′intron fragment as listed in Table 19 (e.g., both representing fragmentswith the L9a-8 permutation site).

TABLE 19 Anabaena permutation site 3' intron fragment sequences.Permutation SEQ ID NO site Sequence 112 L2-1 ACGGACTTAAATAATTGAGCCTTAAA113 L2-2 ACGGACTTAAATAATTGAGCCTTAAAG 114 L2-3ACGGACTTAAATAATTGAGCCTTAAAGA 115 L5-1ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTA 116 L5-2ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAG 117 L5-3ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGT 118 L5-4ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT 119 L5-5ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTA 120 L6-1ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT A 121 L6-2ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AA 122 L6-3ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AAC 123 L6-4ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AACA 124 L6-5ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AACAA 125 L6-6ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AACAAT 126 L6-7ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AACAATA 127 L6-8ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AACAATAG 128 L6-9ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTT AACAATAGA 129 L8-1ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACT CGACGGGAGCTACCCTAACGT 130 L8-2ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACT CGACGGGAGCTACCCTAACGTC 131L8-3 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACT CGACGGGAGCTACCCTAACGTCA 132L8-4 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACT CGACGGGAGCTACCCTAACGTCAA 133L8-5 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACT CGACGGGAGCTACCCTAACGTCAAG 134L9a-1 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAG AGTCCAATTCTCAAAGCC 135 L9a-2ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAG AGTCCAATTCTCAAAGCCA 136 L9a-3ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAG AGTCCAATTCTCAAAGCCAA 137 L9a-4ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAG AGTCCAATTCTCAAAGCCAAT 138L9a-5 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAG AGTCCAATTCTCAAAGCCAATA 139L9-1 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGC 140 L9-2ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCG 141 L9-3ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGA 142 L9-4ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAA 143 L9-5ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAA 144 L9-6ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAG 145 L9-7ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGC 146 L9-8ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCA 147 L9-9ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCA A 148 L9a-6ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAG AGTCCAATTCTCAAAGCCAATAG 149L9a-7 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAG AGTCCAATTCTCAAAGCCAATAGGC 150L9a-8 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAG AGTCCAATTCTCAAAGCCAATAGGCA

In some embodiments, a 3′ intron fragment is a fragment having asequence listed in Table 19. In some embodiments, a construct containinga 3′ intron fragment listed in Table 19 will contain a corresponding 5′intron fragment as listed in Table 18 (e.g., both representing fragmentswith the L9a-8 permutation site).

TABLE 20 Non-anabaena permutation site 5′ intron fragment sequences.SEQ ID NO Intron Sequence 151 Azop1tgcgccgatgaaggtgtagagactagacggcacccacctaaggcaaacgctatggtgaaggcatagtccagggagtggcgaaagtcacacaaaccggaatccgt 152 Azop2ccgggcgtatggcaacgccgagccaagcttcggcgcctgcgccgatgaaggtgtagagactagacggcacccacctaaggcaaacgctatggtgaaggcatagtccagggagtggcgaaagtcacacaaaccggaatccgt 153 Azop3acggcacccacctaaggcaaacgctatggtgaaggcatagtccagggagtggcgaaagtcacacaaaccggaatccgt 154 Azop4acgctatggtgaaggcatagtccagggagtggcgaaagtcacacaaaccggaatccgt 155 S7951p1attaaagttatagaattatcagagaatgatatagtccaagccttatggtaacatgagggcacttgaccctggtag 156 Twortp1aagatgtaggcaatcctgagctaagctcttagtaataagagaaagtgcaacgactattccgataggaagtagggtcaagtgactcgaaatggggattaccattctagggtagtgatatagtctgaacatatatggaaacatatagaaggataggagtaacgaacctattcgtaacataattgaacttttagttat 157 Twortp2taataagagaaagtgcaacgactattccgataggaagtagggtcaagtgactcgaaatggggattaccatctagggtagtgatatagtctgaacatatatggaaacatatagaaggataggagtaacgaacctattcgtaacataattgaacttttagttat 158 Twortp3taggaagtagggtcaagtgactcgaaatggggattacccttctagggtagtgatatagtctgaacatatatggaaacatatagaaggataggagtaacgaacctattcgtaacataattgaacttttagttat 159 Twortp4ctagggtagtgatatagtctgaacatatatggaaacatatagaaggataggagtaacgaacctattcgtaacataattgaacttttagttat 160 LSUp1agttaataaagatgatgaaatagtctgaaccattttgagaaaagtggaaataaaagaaaatcttttatgataacataaattgaacaggctaa 161 Phip1caaagactgatgatatagtccgacactcctagtaataggagaatacagaaaggatgaaatcc 162Nostocagtcgagggtaaagggagagtccaattctcaaagcctattggcagtagcgaaagctgcgggagaatgaaaatccgt 163 Nostocagccgagggtaaagggagagtccaattctcaaagccaataggcagtagcgaaagctgcgggagaatgaaaatccgt 164 Nodulariaagccgagggtaaagggagagtccaattctcaaagccgaaggttattaaaacctggcagcagtgaaagctgcgggagaatgaaaatccgt 165 Pleurocapsaagctgagggtaaagagagagtccaattctcaaagccagcagatggcagtagcgaaagctgcgggagaatgaaaatccgt 166 Planktothrixagccgagggtaaagagagagtccaattctcaaagccaattggtagtagcgaaagctacgggagaatgaaaatccgt

In some embodiments, a 5′ intron fragment is a fragment having asequence listed in Table 20. A construct containing a 5′ intron fragmentlisted in Table 20 will contain a corresponding 3′ intron fragment inTable 21 (e.g., both representing fragments with the Azop1 intron).

TABLE 21 Non-anabaena permutation site 3′ intron fragment sequences.SEQ ID NO Intron Sequence 167 Azop1gcggactcatatttcgatgtgccttgcgccgggaaaccacgcaagggatggtgtcaaattcggcgaaacctaagcgcccgcccgggcgtatggcaacgccgagccaagcttcggcgcc 168 Azop2gcggactcatatttcgatgtgccttgcgccgggaaaccacgcaagggatggtgtcaaattcggcgaaacctaagcgcccgc 169 Azop3gcggactcatatttcgatgtgccttgcgccgggaaaccacgcaagggatggtgtcaaattcggcgaaacctaagcgcccgcccgggcgtatggcaacgccgagccaagcttcggcgcctgcgccgatgaaggtgtagagactag 170 Azop4gcggactcatatttcgatgtgccttgcgccgggaaaccacgcaagggatggtgtcaaattcggcgaaacctaagcgcccgcccgggcgtatggcaacgccgagccaagcttcggcgcctgcgccgatgaaggtgtagagactagacggcacccacctaaggcaa 171 S795p1aggattagatactacactaagtgtcccccagactggtgacagtctggtgtgcatccagctatatcggtgaaaccccattggggtaataccgagggaagctatattatatatatattaataaatagccccgtagagactatgtaggtaaggagatagaagatgataaaatcaaaatcatc 172 Twortp1actactgaaagcataaataattgtgcctttatacagtaatgtatatcgaaaaatcctctaattcagggaacacctaaacaaact 173 Twortp2actactgaaagcataaataattgtgcctttatacagtaatgtatatcgaaaaatcctctaattcagggaacacctaaacaaactaagatgtaggcaatcctgagctaagctcttag 174 Twortp3actactgaaagcataaataattgtgcctttatacagtaatgtatatcgaaaaatcctctaattcagggaacacctaaacaaactaagatgtaggcaatcctgagctaagctcttagtaataagagaaagtgcaacgactattccga 175 Twortp4actactgaaagcataaataattgtgcctttatacagtaatgtatatcgaaaaatcctctaattcagggaacacctaaacaaactaagatgtaggcaatcctgagctaagctcttagtaataagagaaagtgcaacgactattccgataggaagtagggtcaagtgactcgaaatggggattaccctt 176 LSUp1cgctagggatttataactgtgagtcctccaatattataaaatgttggtaatatattgggtaaatttcaaagacaacttttctccacgtcaggatatagtgtatttgaagcgaaacttattttagcagtgaaaaagcaaataaggacgttcaacgactaaaaggtgagtattgctaacaataatccttttttttaatgcccaacatctttattaact177 Phip1gtgggtgcataaactatttcattgtgcacattaaatctggtgaactcggtgaaaccctaatggggcaataccgagccaagccatagggaggatatatgagaggcaagaagttaattcttgaggccactgagactggctgtatcatccctacgtcacacaaacttaatgccgatggttatttcagaaagaaaaccaatggcgtcttagagatgtatcacagaacggtgtggaaggagcataacggagacatacctgatggcttcgagatagaccataagtgtcgcaatagggcttgctgtaatatagagcatttacagatgcttgagggtacagcccacactgttaagaccaatcgtgaacgctacgcagacagaaaggaaacagctagggaatactggctggagactggatgtaccggcctagcactcggtgagaagtttggtgtgtcgttctcttctgcttgtaagtggattagagaatggaaggcgtagagactatccgaaaggagtagggccgagggtgagactccctcgtaacccgaagcgccagacagtcaact 178Nostocacggacttaagtaattgagccttaaagaagaaattctttaagtggcagctctcaaactcagggaaacctaaatctgttcacagacaaggcaatcctgagccaagccgaaagagtcatgagtgctgagtagtgagtaaaataaaagctcacaactcagaggttgtaactctaagctagtcggaaggtgcagagactcgacgggagctaccctaacgtaa 179 Nostocacggacttaaactgaattgagccttagagaagaaattctttaagtgtcagctctcaaactcagggaaacctaaatctgttgacagacaaggcaatcctgagccaagccgagaactctaagttattcggaaggtgcagagactcgacgggagctaccctaacgtca 180 Nodulariaacggacttagaaaactgagccttgatcgagaaatctttcaagtggaagctctcaaattcagggaaacctaaatctgtttacagatatggcaatcctgagccaagccgaaacaagtcctgagtgttaaagctcataactcatcggaaggtgcagagactcgacgggagctaccctaacgtta 181 Pleurocapsaacggacttaaaaaaattgagccttggcagagaaatctgtcatgcgaacgctctcaaattcagggaaacctaagtctggcaacagatatggcaatcctgagccaagccttaatcaaggaaaaaaacatttttaccttttaccttgaaaggaaggtgcagagactcaacgggagctaccctaacaggtca 182 Planktothrixacggacttaaagataaattgagccttgaggcgagaaatctctcaagtgtaagctgtcaaattcagggaaacctaaatctgtaaattcagacaaggcaatcctgagccaagcctaggggtattagaaatgagggagtttccccaatctaagatcaatacctaggaaggtgcagagactcgacgggagctaccctaacgtta

In some embodiments, a 3′ intron fragment is a fragment having asequence listed in Table 21. A construct containing a 3′ intron fragmentlisted in Table 21 will contain the corresponding 5′ intron fragment aslisted in Table 20 (e.g., both representing fragments with the Azop1intron).

TABLE 22 Spacer and Anabaena 5′ intron fragment sequences. SEQ ID NOSpacer Sequence 183 T25 L10agtatataagaaacaaaccacTAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggctcgcagc 184 T25L20ctgaaattatacttatactcaaacaaaccacTAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggctcgcagc 185 T25 L30ctgaaattatacttatactcagtatatgacaaacaaaccacTAGATGACTTACAACTAA (I80-10)TCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAA [Control]GACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggctcgcagc 186 T25 L40catcaacaatatgaaattatacttatactcagtatatgacaaacaaaccacTAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggct cgcagc 187 T25 L50catcaacaatatgaaactatacttatactcagtatatgaagcattatcgcaaacaaaccacTAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAAT CCGTggctcgcagc 188 T50 L10tagcgtcagcaaacaaacaaaTAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggctcgcagc 189 T50 L20atactcatactagcgtcagcaaacaaacaaaTAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggctcgcagc 190 T50 L30gtgtgaagctatactcatactagcgtcagcaaacaaacaaaTAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggctcgcagc 191 T50 L40cctcacctgagtgtgaagctatactcatactagcgtcagcaaacaaacaaaTAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTgg ctcgcagc 192 T50 L50ccgaatgatgcctcacctgagtgtgaagctatactcatactagcgtcagcaaacaaacaaaTAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAA TCCGTggctcgcagc 193 T75 L10cggtgcgagcaaacaaacaaaTAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggctcgcagc 194 T75 L20cgctccgacccagtgcgagcaaacaaacaaaTAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggctcgcagc 195 T25 L30ctgaaattatactAatactcagtatatgacaaacaaaccacTAGATGACTTACAACTAA 1 MMTCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggctcgcagc 196 T25 L30ctgaaaAtatactAatactcaCtatatgacaaacaaaccacTAGATGACTTACAACTA 3 MMATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggctcgcagc 197 T25 L30ctgaTaAtataGtAatactcaCtatatgacaaacaaaccacTAGATGACTTACAACT 5 MMAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggctcgcagc 198  T25 L30ctgaTaAtaAaGtAatacAcaCtataAgacaaacaaaccacTAGATGACTTACAA 8 MMCTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggctcgc agc 199 T25 L30ctgaaattatacttatactctctaagttacaaacaaaccacTAGATGACTTACAACTAATOffTarget 10 CGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggctcgcagc 200 T25 L30ctgaaattatgtgtgttacAtctaagttacaaacaaaccacTAGATGACTTACAACTAA OffTarget 20TCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggctcgcagc 201 T25 L30gagatcggtgtgtgttacAtctaagttacaaacaaaccacTAGATGACTTACAACTAA OffTarget 30TCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggctcgcagc 202 T25 L30ctgaaattatacttatactcagtatatgacaaacaaaccacTAGATGACTTACAACTAA I25-10TCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTgattaaacag 203 T25 L30ctgaaattatacttatactcagtatatgacaaacaaaccacTAGATGACTTACAACTAA I25-20TCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTgattcacaatataaa ttacg 204 T25 L30ctgaaattatacttatactcagtatatgacaaacaaaccacTAGATGACTTACAACTAA I50-10TCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggatcatagc 205 T25 L30ctgaaattatacttatactcagtatatgacaaacaaaccacTAGATGACTTACAACTAA I50-20TCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggatcgcagcataa tatccg 206 T25 L30ctgaaattatacttatactcagtatatgacaaacaaaccacTAGATGACTTACAACTAA I80-20TCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggctcgcagcgcg cctaccg 207 T25 L30ctgaaattatacttatactcagtatatgacaaacaaaccacTAGATGACTTACAACTAA I80-20x2TCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggctcgcagcgcgcctaccgaaagccggcgtcgacgttagcgc 208 T25 L30ctgaaattatacttatactcagtatatgacaaacaaaccacTAGATGACTTACAACTAA I50-20x2TCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggatcgcagcataatatccgaaacgaggatacaagtgacatgc 209 T25 L30ctgaaattatacttatactcagtatatgacaaacaaaccacTAGATGACTTACAACTAA I25-20x2TCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTgattcacaatctaaattacgaaacgataaatgataactctaac 210 T0 L0aaacaaaccacTAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAA GAGAATGAAAATCCGTggctcgcagc211 T100 L5 cgggcaaacaaacaaaTAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggctcgcagc 212 T75 L30cgctccgacgagatccggccagtgcgagcaaacaaacaaaTAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggctcgcagc 213 T0 L0aaaacaaaccacGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCC GTggctcgcagc 214 T25 L10aagtatataagaaacaaaccacGGCAGTAGCGAAAGCTGCAAGAGAATGA AAATCCGTggctcgcagc 215T25 L20a ctgaaattatacttatactcaaacaaaccacGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggctcgcagc 216 T25 L30actgaaattatacttatactcagtatatgacaaacaaaccacGGCAGTAGCGAAAGCTGC (I80-10)AAGAGAATGAAAATCCGTggctcgcagc [Control] 217 T50 L10atagcgtcagcaaacaaacaaaGGCAGTAGCGAAAGCTGCAAGAGAATGA AAATCCGTggctcgcagc 218T50 L20a atactcatactagcgtcagcaaacaaacaaaGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggctcgcagc 219 T50 L30agtgtgaagctatactcatactagcgtcagcaaacaaacaaaGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggctcgcagc 220 T75 L10acggtgcgagcaaacaaacaaaGGCAGTAGCGAAAGCTGCAAGAGAATGA AAATCCGTggctcgcagc 221T75 L20a cgctccgacccagtgcgagcaaacaaacaaaGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggctcgcagc 222 T75 L30acgctccgacgagatccggccagtgcgagcaaacaaacaaaGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggctcgcagc 223 T0 L0baaacaaaccacAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCC GTggctcgcagc 224 T25 L10bagtatataagaaacaaaccacAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAA AATCCGTggctcgcagc 225 T25 L20bctgaaattatacttatactcaaacaaaccacAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGA ATGAAAATCCGTggctcgcagc 226T25 L30b ctgaaattatacttatactcagtatatgacaaacaaaccacAAGACGAGGGTAAAGAG(I80-10) AGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGC [Control]AAGAGAATGAAAATCCGTggctcgcagc 227 T50 L10btagcgtcagcaaacaaacaaaAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGA AAATCCGTggctcgcagc 228T50 L20b atactcatactagcgtcagcaaacaaacaaaAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAG AATGAAAATCCGTggctcgcagc 229T50 L30b gtgtgaagctatactcatactagcgtcagcaaacaaacaaaAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTG CAAGAGAATGAAAATCCGTggctcgcagc230 T75 L10b cggtgcgagcaaacaaacaaaAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGA AAATCCGTggctcgcagc 231T75 L20b cgctccgacccagtgcgagcaaacaaacaaaAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGA GAATGAAAATCCGTggctcgcagc 232T75 L30b cgctccgacgagatccggccagtgcgagcaaacaaacaaaAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggctcgcagc 233 T25 L30ctgaaattatacttatactcagtatatgacaaacaaaccacTAGATGACTTACAACTAA I0-0TCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGT 234 T25 L30actgaaattatacttatactcagtatatgacaaacaaaccacGGCAGTAGCGAAAGCTGC I0-0AAGAGAATGAAAATCCGT 235 T25 L30actgaaattatacttatactcagtatatgacaaacaaaccacGGCAGTAGCGAAAGCTGC I25-10AAGAGAATGAAAATCCGTgattaaacag 236 T25 L30actgaaattatacttatactcagtatatgacaaacaaaccacGGCAGTAGCGAAAGCTGC I25-20AAGAGAATGAAAATCCGTgattcacaatataaattacg 237 T25 L30actgaaattatacttatactcagtatatgacaaacaaaccacGGCAGTAGCGAAAGCTGC I50-10AAGAGAATGAAAATCCGTggatcatagc 238 T25 L30actgaaattatacttatactcagtatatgacaaacaaaccacGGCAGTAGCGAAAGCTGC I50-20AAGAGAATGAAAATCCGTggatcgcagcataatatccg 239 T25 L30actgaaattatacttatactcagtatatgacaaacaaaccacGGCAGTAGCGAAAGCTGC I80-20AAGAGAATGAAAATCCGTggctcgcagcgcgcctaccg 240 T25 L30bctgaaattatacttatactcagtatatgacaaacaaaccacAAGACGAGGGTAAAGAG I0-0AGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGC AAGAGAATGAAAATCCGT 241T25 L30b ctgaaattatacttatactcagtatatgacaaacaaaccacAAGACGAGGGTAAAGAGI25-10 AGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTgattaaacag 242 T25 L30bctgaaattatacttatactcagtatatgacaaacaaaccacAAGACGAGGGTAAAGAG I25-20AGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTgattcacaatataaattacg 243 T25 L30bctgaaattatacttatactcagtatatgacaaacaaaccacAAGACGAGGGTAAAGAG I50-10AGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGC AAGAGAATGAAAATCCGTggatcatagc244 T25 L30b ctgaaattatacttatactcagtatatgacaaacaaaccacAAGACGAGGGTAAAGAGI50-20 AGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggatcgcagcataatatccg 245 T25 L30bctgaaattatacttatactcagtatatgacaaacaaaccacAAGACGAGGGTAAAGAG I80-20AGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTggctcgcagcgcgcctaccg

In some embodiments, a spacer and 5′ intron fragment are spacers andfragments having sequences as listed in Table 22.

TABLE 23 Spacer and Anabaena 3′ intron fragment sequences. SEQ ID NOSpacer Sequence 246 T25 L10gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAAcacaaacacaacttatatact 247 T25 L20gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAAcacaaacacaagagtataagtataatttcag 248 T25 L30gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC (I80-10)TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT [Control]ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAAcacaaacacaagtcatatactgagtataagtataatttcag 249 T25 L40gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAAcacaaacacaagtcatatactgagtataagtataatttcatattgttgatg 250T25 L50 gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAAcacaaacacaagcgataatgcttcatatactgagtataagtatagatcatattg ttgatg251 T50 L10 gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAAaacaaaaacaagctgacgcta 252 T50 L20gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAAaacaaaaacaagctgacgctagtatgagtat 253 T50 L30gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAAaacaaaaacaagctgacgctagtatgagtatagcttcacac 254 T50 L40gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAAaacaaaaacaagctgacgctagtatgagtatagcttcacactcaggtgagg 255T50 L50 gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAAaacaaaaacaagctgacgctagtatgagtatagcttcacactcaggtgaggc atcattcgg256 T75 L10 gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAAaacaaaaacaagctcgcaccg 257 T75 L20gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAAaacaaaaacaagctcgcactgggtcggagcg 258 T25 L30gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC 1 MMTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAAcacaaacacaagtcatatactgagtataagtataatttcag 259 T25 L30gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC 3 MMTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAAcacaaacacaagtcatatactgagtataagtataatttcag 260 T25 L30gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC 5 MMTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAAcacaaacacaagtcatatactgagtataagtataatttcag 261 T25 L30gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC 8 MMTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAAcacaaacacaagtcatatactgagtataagtataatttcag 262 T25 L30gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC OffTarget 10TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAAcacaaacacaagtaacttagagagtataagtataatttcag 263 T25 L30gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC OffTarget 20TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAAcacaaacacaagtaacttagaTgtaacacacataatttcag 264 T25 L30gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC OffTarget 30TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAAcacaaacacaagtaacttagaTgtaacacacaccgatcaac 265 T25 L30ctgtttaatcACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCT I25-10TTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAAcacaaacacaagtcatatactgagtataagtataatttcag 266 T25 L30cgtaatttatattgtgaatcACGGACTTAAATAATTGAGCCTTAAAGAAGA I25-20AATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAAcacaaacacaagtcatatactgagtataagtataatttcag 267 T25 L30gctatgatccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC I50-10TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAAcacaaacacaagtcatatactgagtataagtataatttcag 268 T25 L30cggatattatgctgcgatccACGGACTTAAATAATTGAGCCTTAAAGAAG I50-20AAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAAcaeaaacacaagtcatatactgagtataagtataatttcag 269 T25 L30cggtaggcgctgctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAA I80-20GAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAAcaeaaacacaagtcatatactgagtataagtataatttcag 270 T25 L30gcgctaacgtcgacgccggcaaacggtaggcgcgctgcgagccACGGACTTAAATAA I80-20x2TTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCrCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAAcacaaacacaagtcatatactgagtataagtataatttcag 271 T25 L30gcatgtcacttgtatcctcgaaacggatattatgctgcgatccACGGACTTAAATAATTG I50-20x2AGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAAcacaaacacaagtcatatac tgagtataagtataatttcag272 T25 L30gttagagttatcatttatcgaaacgtaatttagattgtgaatcACGGACTTAAATAATTGA I25-20x2GCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAAcacaaacacaagtcatatactg agtataagtataatttcag273 T0 L0 gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAAcacaaacacaa 274T100 L5 gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGT AAGTTAACAAaacaaaaacaagcccg275 T75 L30 gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAAaacaaaaacaagctcgcactggccggaagctcgtcggagcg 276 T0 L0agctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAcacaaacacaa 277 T25 L10agctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAcacaaacacaacttatatact 278 T25 L20agctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAcacaaacacaagagtataagtataatttc ag 279 T25 L30agctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC (I80-10)TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT [Control]ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAcacaaacacaagtcatatactgagtataa gtataatttcag 280T50 L10a gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAaacaaaaacaagctgacgcta 281 T50 L20agctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAaacaaaaacaagctgacgctagtatga gtat 282 T50 L30agctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAaacaaaaacaagctgacgctagtatga gtatagcttcacac 283T75 L10a gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAaacaaaaacaagctcgcaccg 284 T75 L20agctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAaacaaaaacaagctcgcactgggtcgg agcg 285 T75 L30agctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAaacaaaaacaagctcgcactggccgga agctcgtcggagcg 286T0 L0b gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCcacaaacacaa 287 T25 L10bgctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCcacaaacacaacttatatact 288 T25 L20bgctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCcacaaacacaagagtataagtataatt tcag 289 T25 L30bgctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC (I80-10)TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTT [Control]ATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCcacaaacacaagtcatatactgagtat aagtataatttcag 290T50 L10b gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCaacaaaaacaagctgacgcta 291 T50 L20bgctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCaacaaaaacaagctgacgctagtatg agtat 292 T50 L30bgctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCaacaaaaacaagctgacgctagtatg agtatagcttcacac 293T75 L10b gctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCaacaaaaacaagctcgcaccg 294 T75 L20bgctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCaacaaaaacaagctcgcactgggtcg gagcg 295 T75 L30bgctgcgagccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCaacaaaaacaagctcgcactggccg gaagctcgtcggagcg 296T25 L30 ACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG I0-0TGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAAcacaaacacaagtcatatactgagtataagtataatttcag 297 T25 L30aACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAG I0-0TGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAcacaaacacaagtcatatactgagtataagtataat ttcag 298T25 L30a ctgataatcACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCT I25-10TTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAcacaaacacaagtcatatactgagtataa gtataatttcag 299T25 L30a cgtaatttatattgtgaatcACGGACTTAAATAATTGAGCCTTAAAGAAGA I25-20AATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAcacaaacacaagtcatatac tgagtataagtataatttcag300 T25 L30a gctatgatccACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTC I50-10TTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGTTAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAcacaaacacaagtcatatactgagtataa gtataatttcag

In some embodiments, a spacer and 3′ intron fragment is a spacer andintron fragment having sequences as listed in Table 23.

TABLE 24 CAR sequences SEQ ID NO CAR Sequence 309 FMC63-4-ATGCTGCTGCTGGTCACATCTCTGCTGCTGTGCGAGCTGCCCC 1BBATCCTGCCTTTCTGCTGATCCCCGACATCCAGATGACCCAGACCACAAGCAGCCTGTCTGCCAGCCTGGGCGATAGAGTGACCATCAGCTGTAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAAAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGACTGCACAGCGGCGTGCCAAGCAGATTTTCTGGCAGCGGCTCTGGCACCGACTACAGCCTGACAATCAGCAACCTGGAACAAGAGGATATCGCTACCTACTTCTGCCAGCAAGGCAACACCCTGCCTTACACCTTTGGCGGAGGCACCAAGCTGGAAATCACCGGCTCTACAAGCGGCAGCGGCAAACCTGGATCTGGCGAGGGATCTACCAAGGGCGAAGTGAAACTGCAAGAGTCTGGCCCTGGACTGGTGGCCCCATCTCAGTCTCTGAGCGTGACCTGTACAGTCAGCGGAGTGTCCCTGCCTGATTACGGCGTGTCCTGGATCAGACAGCCTCCTCGGAAAGGCCTGGAATGGCTGGGAGTGATCTGGGGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTATTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGATTATTGGGGCCAGGGCACCAGCGTGACCGTTTCTTCTGCCGCCGCTATCGAAGTGATGTACCCTCCTCCTTACCTGGACAACGAGAAGTCCAACGGCACCATCATCCACGTGAAGGGCAAGCACCTGTGTCCTTCTCCACTGTTCCCCGGACCTAGCAAGCCTTTCTGGGTGCTCGTTGTTGTTGGCGGCGTGCTGGCCTGTTACAGCCTGCTGGTTACCGTGGCCTTCATCATCTTTTGGGTCAAGAGAGGCCGGAAGAAACTTCTTTATATATTCAAGCAGCCCTTTATGCGACCCGTTCAGACTACCCAAGAGGAAGATGGATGCAGTTGCCGCTTTCCAGAAGAGGAGGAGGGCGGGTGCGAACTGtaa 310 FMC63-CD28ATGCTGCTGCTGGTCACATCTCTGCTGCTGTGCGAGCTGCCCCATCCTGCCTTTCTGCTGATCCCCGACATCCAGATGACCCAGACCACAAGCAGCCTGTCTGCCAGCCTGGGCGATAGAGTGACCATCAGCTGTAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAAAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGACTGCACAGCGGCGTGCCAAGCAGATTTTCTGGCAGCGGCTCTGGCACCGACTACAGCCTGACAATCAGCAACCTGGAACAAGAGGATATCGCTACCTACTTCTGCCAGCAAGGCAACACCCTGCCTTACACCTTTGGCGGAGGCACCAAGCTGGAAATCACCGGCTCTACAAGCGGCAGCGGCAAACCTGGATCTGGCGAGGGATCTACCAAGGGCGAAGTGAAACTGCAAGAGTCTGGCCCTGGACTGGTGGCCCCATCTCAGTCTCTGAGCGTGACCTGTACAGTCAGCGGAGTGTCCCTGCCTGATTACGGCGTGTCCTGGATCAGACAGCCTCCTCGGAAAGGCCTGGAATGGCTGGGAGTGATCTGGGGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTATTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGATTATTGGGGCCAGGGCACCAGCGTGACCGTTTCTTCTGCCGCCGCTATCGAAGTGATGTACCCTCCTCCTTACCTGGACAACGAGAAGTCCAACGGCACCATCATCCACGTGAAGGGCAAGCACCTGTGTCCTTCTCCACTGTTCCCCGGACCTAGCAAGCCTTTCTGGGTGCTCGTTGTTGTTGGCGGCGTGCTGGCCTGTTACAGCCTGCTGGTTACCGTGGCCTTCATCATCTTTTGGGTCCGAAGCAAGCGGAGCCGGCTGCTGCACTCCGACTACATGAACATGACCCCTAGACGGCCCGGACCAACCAGAAAGCACTACCAGCCTTACGCTCCTCCTAGAGACTTCGCCGCCTACCGGTCCtaa 311 FMC63-ATGCTGCTGCTGGTCACATCTCTGCTGCTGTGCGAGCTGCCCC CD28-zetaATCCTGCCTTTCTGCTGATCCCCGACATCCAGATGACCCAGACCACAAGCAGCCTGTCTGCCAGCCTGGGCGATAGAGTGACCATCAGCTGTAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAAAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGACTGCACAGCGGCGTGCCAAGCAGATTTTCTGGCAGCGGCTCTGGCACCGACTACAGCCTGACAATCAGCAACCTGGAACAAGAGGATATCGCTACCTACTTCTGCCAGCAAGGCAACACCCTGCCTTACACCTTTGGCGGAGGCACCAAGCTGGAAATCACCGGCTCTACAAGCGGCAGCGGCAAACCTGGATCTGGCGAGGGATCTACCAAGGGCGAAGTGAAACTGCAAGAGTCTGGCCCTGGACTGGTGGCCCCATCTCAGTCTCTGAGCGTGACCTGTACAGTCAGCGGAGTGTCCCTGCCTGATTACGGCGTGTCCTGGATCAGACAGCCTCCTCGGAAAGGCCTGGAATGGCTGGGAGTGATCTGGGGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTATTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGATTATTGGGGCCAGGGCACCAGCGTGACCGTTTCTTCTGCCGCCGCTATCGAAGTGATGTACCCTCCTCCTTACCTGGACAACGAGAAGTCCAACGGCACCATCATCCACGTGAAGGGCAAGCACCTGTGTCCTTCTCCACTGTTCCCCGGACCTAGCAAGCCTTTCTGGGTGCTCGTTGTTGTTGGCGGCGTGCTGGCCTGTTACAGCCTGCTGGTTACCGTGGCCTTCATCATCTTTTGGGTCCGAAGCAAGCGGAGCCGGCTGCTGCACTCCGACTACATGAACATGACCCCTAGACGGCCCGGACCAACCAGAAAGCACTACCAGCCTTACGCTCCTCCTAGAGACTTCGCCGCCTACCGGTCCAGAGTGAAGTTCAGCAGATCCGCCGATGCTCCCGCCTATCAGCAGGGCCAAAACCAGCTGTACAACGAGCTGAACCTGGGGAGAAGAGAAGAGTACGACGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAAATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAGGGCCTGTATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTACCAGGGACTGAGCACCGCCACCAAGGATACCTATGACGCCCTGCACATGCAGGCCCTGCCTCCAAGAtaa 312 FMC63-zetaATGCTGCTGCTGGTCACATCTCTGCTGCTGTGCGAGCTGCCCCATCCTGCCTTTCTGCTGATCCCCGACATCCAGATGACCCAGACCACAAGCAGCCTGTCTGCCAGCCTGGGCGATAGAGTGACCATCAGCTGTAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAAAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGACTGCACAGCGGCGTGCCAAGCAGATTTTCTGGCAGCGGCTCTGGCACCGACTACAGCCTGACAATCAGCAACCTGGAACAAGAGGATATCGCTACCTACTTCTGCCAGCAAGGCAACACCCTGCCTTACACCTTTGGCGGAGGCACCAAGCTGGAAATCACCGGCTCTACAAGCGGCAGCGGCAAACCTGGATCTGGCGAGGGATCTACCAAGGGCGAAGTGAAACTGCAAGAGTCTGGCCCTGGACTGGTGGCCCCATCTCAGTCTCTGAGCGTGACCTGTACAGTCAGCGGAGTGTCCCTGCCTGATTACGGCGTGTCCTGGATCAGACAGCCTCCTCGGAAAGGCCTGGAATGGCTGGGAGTGATCTGGGGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTATTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGATTATTGGGGCCAGGGCACCAGCGTGACCGTTTCTTCTGCCGCCGCTATCGAAGTGATGTACCCTCCTCCTTACCTGGACAACGAGAAGTCCAACGGCACCATCATCCACGTGAAGGGCAAGCACCTGTGTCCTTCTCCACTGTTCCCCGGACCTAGCAAGCCTTTCTGGGTGCTCGTTGTTGTTGGCGGCGTGCTGGCCTGTTACAGCCTGCTGGTTACCGTGGCCTTCATCATCTTTTGGGTCAGAGTGAAGTTCAGCAGATCCGCCGATGCTCCCGCCTATCAGCAGGGCCAAAACCAGCTGTACAACGAGCTGAACCTGGGGAGAAGAGAAGAGTACGACGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAAATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAGGGCCTGTATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTACCAGGGACTGAGCACCGCCACCAAGGATACCTATGACGCCCTGCACATGCAGGCCCTGCCTCCAAGAtaa 313 CircKymriah-ATGGCTCTCCCGGTCACAGCCCTTCTCCTGCCCCTGGCACTCT Q388TGCTGCATGCGGCACGACCCGACATCCAGATGACCCAGACCACAAGCAGCCTGTCTGCCAGCCTGGGCGATAGAGTGACCATCAGCTGTAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAAAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGACTGCACAGCGGCGTGCCAAGCAGATTTTCTGGCAGCGGCTCTGGCACCGACTACAGCCTGACAATCAGCAACCTGGAACAAGAGGATATCGCTACCTACTTCTGCCAGCAAGGCAACACCCTGCCTTACACCTTTGGCGGAGGCACCAAGCTGGAAATCACCGGTGGAGGTGGTTCTGGCGGAGGGGGATCTGGTGGAGGCGGTTCAGAAGTGAAACTGCAAGAGTCTGGCCCTGGACTGGTGGCCCCATCTCAGTCTCTGAGCGTGACCTGTACAGTCAGCGGAGTGTCCCTGCCTGATTACGGCGTGTCCTGGATCAGACAGCCTCCTCGGAAAGGCCTGGAATGGCTGGGAGTGATCTGGGGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTATTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGATTATTGGGGCCAGGGCACCAGCGTGACCGTTTCTTCTACCACAACGCCCGCCCCGCGACCGCCTACTCCCGCTCCCACAATTGCATCACAACCCCTGTCTTTGAGACCCGAAGCTTGTCGACCAGCTGCCGGTGGCGCGGTTCACACGCGGGGGCTCGATTTCGCCTGTGATATATATATATGGGCCCCATTGGCTGGAACATGCGGAGTATTGCTTCTGAGCCTGGTGATTACCCTCTACTGTAAGAGAGGCCGGAAGAAACTTCTTTATATATTCAAGCAGCCCTTTATGCGACCCGTTCAGACTACCCAAGAGGAAGATGGATGCAGTTGCCGCTTTCCAGAAGAGGAGGAGGGCGGGTGCGAACTGAGAGTGAAGTTCAGCAGATCCGCCGATGCTCCCGCCTATCAGCAGGGCCAAAACCAGCTGTACAACGAGCTGAACCTGGGGAGAAGAGAAGAGTACGACGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAAATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAGGGCCTGTATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTACCAGGGACTGAGCACCGCCACCAAGGATACCTATGACGCCCTGCACATGCAGGCCCTGCCTCCAAGAtaa 314 CircKymriah-ATGGCTCTCCCGGTCACAGCCCTTCTCCTGCCCCTGGCACTCT K388TGCTGCATGCGGCACGACCCGACATCCAGATGACCCAGACCACAAGCAGCCTGTCTGCCAGCCTGGGCGATAGAGTGACCATCAGCTGTAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAAAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGACTGCACAGCGGCGTGCCAAGCAGATTTTCTGGCAGCGGCTCTGGCACCGACTACAGCCTGACAATCAGCAACCTGGAACAAGAGGATATCGCTACCTACTTCTGCCAGCAAGGCAACACCCTGCCTTACACCTTTGGCGGAGGCACCAAGCTGGAAATCACCGGTGGAGGTGGTTCTGGCGGAGGGGGATCTGGTGGAGGCGGTTCAGAAGTGAAACTGCAAGAGTCTGGCCCTGGACTGGTGGCCCCATCTCAGTCTCTGAGCGTGACCTGTACAGTCAGCGGAGTGTCCCTGCCTGATTACGGCGTGTCCTGGATCAGACAGCCTCCTCGGAAAGGCCTGGAATGGCTGGGAGTGATCTGGGGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTATTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGATTATTGGGGCCAGGGCACCAGCGTGACCGTTTCTTCTACCACAACGCCCGCCCCGCGACCGCCTACTCCCGCTCCCACAATTGCATCACAACCCCTGTCTTTGAGACCCGAAGCTTGTCGACCAGCTGCCGGTGGCGCGGTTCACACGCGGGGGCTCGATTTCGCCTGTGATATATATATATGGGCCCCATTGGCTGGAACATGCGGAGTATTGCTTCTGAGCCTGGTGATTACCCTCTACTGTAAGAGAGGCCGGAAGAAACTTCTTTATATATTCAAGCAGCCCTTTATGCGACCCGTTCAGACTACCCAAGAGGAAGATGGATGCAGTTGCCGCTTTCCAGAAGAGGAGGAGGGCGGGTGCGAACTGAGAGTGAAGTTCAGCAGATCCGCCGATGCTCCCGCCTATAAGCAGGGCCAAAACCAGCTGTACAACGAGCTGAACCTGGGGAGAAGAGAAGAGTACGACGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAAATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAGGGCCTGTATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTACCAGGGACTGAGCACCGCCACCAAGGATACCTATGACGCCCTGCACATGCAGGCCCTGCCTCCAAGAtaa 315 CircM971-ATGCTGCTGCTGGTCACATCTCTGCTGCTGTGCGAGCTGCCCC CD22ATCCTGCCTTTCTGCTGATCCCCCAGGTTCAACTCCAGCAGTCTGGTCCCGGCCTCGTTAAACCAAGCCAGACTTTGTCTCTTACCTGTGCTATCAGTGGCGATAGCGTGTCTAGTAATTCAGCCGCATGGAACTGGATCCGACAATCACCGAGTAGGGGACTTGAATGGCTGGGTAGAACCTATTACCGGTCCAAATGGTACAATGACTATGCAGTGTCTGTAAAAAGCAGGATCACGATCAACCCTGATACGTCTAAAAACCAGTTTTCTCTGCAACTTAATAGTGTGACCCCTGAAGACACCGCTGTGTATTACTGTGCACGGGAGGTTACCGGTGATCTTGAAGATGCTTTTGATATATGGGGCCAAGGTACGATGGTCACGGTGTCTAGTgggggaggcggcagcGACATACAGATGACGCAGAGCCCATCCAGTCTCTCCGCGTCTGTTGGTGACAGAGTGACTATTACATGTAGGGCGTCTCAGACCATTTGGTCTTACCTCAATTGGTATCAACAGCGACCAGGCAAAGCACCGAACTTGCTCATTTACGCTGCCAGCTCACTCCAAAGTGGTGTGCCGTCCAGATTTAGTGGTAGGGGCAGTGGCACTGATTTCACTCTGACTATTTCAAGTCTTCAAGCTGAGGATTTTGCCACATACTACTGCCAGCAAAGTTACTCAATACCTCAGACTTTTGGACAGGGGACAAAATTGGAGATTAAAtccggaACCACAACGCCCGCCCCGCGACCGCCTACTCCCGCTCCCACAATTGCATCACAACCCCTGTCTTTGAGACCCGAAGCTTGTCGACCAGCTGCCGGTGGCGCGGTTCACACGCGGGGGCTCGATTTCGCCTGTGATATATATATATGGGCCCCATTGGCTGGAACATGCGGAGTATTGCTTCTGAGCCTGGTGATTACCCTCTACTGTAAGAGAGGCCGGAAGAAACTTCTTTATATATTCAAGCAGCCCTTTATGCGACCCGTTCAGACTACCCAAGAGGAAGATGGATGCAGTTGCCGCTTTCCAGAAGAGGAGGAGGGCGGGTGCGAACTGAGAGTGAAGTTCAGCAGATCCGCCGATGCTCCCGCCTATAAGCAGGGCCAAAACCAGCTGTACAACGAGCTGAACCTGGGGAGAAGAGAAGAGTACGACGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAAATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAGGGCCTGTATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTACCAGGGACTGAGCACCGCCACCAAGGATACCTATGACGCCCTGCACATGCAGGCCCT GCCTCCAAGAtaa 316 CircCD19_22ATGCTGCTGCTGGTCACATCTCTGCTGCTGTGCGAGCTGCCCC Bispecific 29ATCCTGCCTTTCTGCTGATCCCCGACATCCAGATGACCCAGACCACAAGCAGCCTGTCTGCCAGCCTGGGCGATAGAGTGACCATCAGCTGTAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAAAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGACTGCACAGCGGCGTGCCAAGCAGATTTTCTGGCAGCGGCTCTGGCACCGACTACAGCCTGACAATCAGCAACCTGGAACAAGAGGATATCGCTACCTACTTCTGCCAGCAAGGCAACACCCTGCCTTACACCTTTGGCGGAGGCACCAAGCTGGAAATCACCggcggcggaggatccCAGGTTCAACTCCAGCAGTCTGGTCCCGGCCTCGTTAAACCAAGCCAGACTTTGTCTCTTACCTGTGCTATCAGTGGCGATAGCGTGTCTAGTAATTCAGCCGCATGGAACTGGATCCGACAATCACCGAGTAGGGGACTTGAATGGCTGGGTAGAACCTATTACCGGTCCAAATGGTACAATGACTATGCAGTGTCTGTAAAAAGCAGGATCACGATCAACCCTGATACGTCTAAAAACCAGTTTTCTCTGCAACTTAATAGTGTGACCCCTGAAGACACCGCTGTGTATTACTGTGCACGGGAGGTTACCGGTGATCTTGAAGATGCTTTTGATATATGGGGCCAAGGTACGATGGTCACGGTGTCTAGTGGCTCTACAAGCGGCAGCGGCAAACCTGGATCTGGCGAGGGATCTACCAAGGGCGACATACAGATGACGCAGAGCCCATCCAGTCTCTCCGCGTCTGTTGGTGACAGAGTGACTATTACATGTAGGGCGTCTCAGACCATTTGGTCTTACCTCAATTGGTATCAACAGCGACCAGGCAAAGCACCGAACTTGCTCATTTACGCTGCCAGCTCACTCCAAAGTGGTGTGCCGTCCAGATTTAGTGGTAGGGGCAGTGGCACTGATTTCACTCTGACTATTTCAAGTCTTCAAGCTGAGGATTTTGCCACATACTACTGCCAGCAAAGTTACTCAATACCTCAGACTTTTGGACAGGGGACAAAATTGGAGATTAAAgggggaggcggcagcGAAGTGAAACTGCAAGAGTCTGGCCCTGGACTGGTGGCCCCATCTCAGTCTCTGAGCGTGACCTGTACAGTCAGCGGAGTGTCCCTGCCTGATTACGGCGTGTCCTGGATCAGACAGCCTCCTCGGAAAGGCCTGGAATGGCTGGGAGTGATCTGGGGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTATTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGATTATTGGGGCCAGGGCACCAGCGTGACCGTTTCTTCTtccggaACCACAACGCCCGCCCCGCGACCGCCTACTCCCGCTCCCACAATTGCATCACAACCCCTGTCTTTGAGACCCGAAGCTTGTCGACCAGCTGCCGGTGGCGCGGTTCACACGCGGGGGCTCGATTTCGCCTGTGATATATATATATGGGCCCCATTGGCTGGAACATGCGGAGTATTGCTTCTGAGCCTGGTGATTACCCTCTACTGTAAGAGAGGCCGGAAGAAACTTCTTTATATATTCAAGCAGCCCTTTATGCGACCCGTTCAGACTACCCAAGAGGAAGATGGATGCAGTTGCCGCTTTCCAGAAGAGGAGGAGGGCGGGTGCGAACTGAGAGTGAAGTTCAGCAGATCCGCCGATGCTCCCGCCTATAAGCAGGGCCAAAACCAGCTGTACAACGAGCTGAACCTGGGGAGAAGAGAAGAGTACGACGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAAATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAGGGCCTGTATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTACCAGGGACTGAGCACCGCCACCAAGGATACCTATGACGCCCTGCACATGCAGGCCCTGCCTCCAAGAtaa 317 CircCD19_22ATGCTGCTGCTGGTCACATCTCTGCTGCTGTGCGAGCTGCCCC Bispecific 30ATCCTGCCTTTCTGCTGATCCCCCAGGTTCAACTCCAGCAGTCTGGTCCCGGCCTCGTTAAACCAAGCCAGACTTTGTCTCTTACCTGTGCTATCAGTGGCGATAGCGTGTCTAGTAATTCAGCCGCATGGAACTGGATCCGACAATCACCGAGTAGGGGACTTGAATGGCTGGGTAGAACCTATTACCGGTCCAAATGGTACAATGACTATGCAGTGTCTGTAAAAAGCAGGATCACGATCAACCCTGATACGTCTAAAAACCAGTTTTCTCTGCAACTTAATAGTGTGACCCCTGAAGACACCGCTGTGTATTACTGTGCACGGGAGGTTACCGGTGATCTTGAAGATGCTTTTGATATATGGGGCCAAGGTACGATGGTCACGGTGTCTAGTgggggaggcggcagcGACATACAGATGACGCAGAGCCCATCCAGTCTCTCCGCGTCTGTTGGTGACAGAGTGACTATTACATGTAGGGCGTCTCAGACCATTTGGTCTTACCTCAATTGGTATCAACAGCGACCAGGCAAAGCACCGAACTTGCTCATTTACGCTGCCAGCTCACTCCAAAGTGGTGTGCCGTCCAGATTTAGTGGTAGGGGCAGTGGCACTGATTTCACTCTGACTATTTCAAGTCTTCAAGCTGAGGATTTTGCCACATACTACTGCCAGCAAAGTTACTCAATACCTCAGACTTTTGGACAGGGGACAAAATTGGAGATTAAAGGGGGAGGCGGATCCGGCGGTGGTGGCTCCGGCGGTGGTGGTTCTGGAGGCGGCGGAAGCGGTGGGGGTGGTAGCGACATCCAGATGACCCAGACCACAAGCAGCCTGTCTGCCAGCCTGGGCGATAGAGTGACCATCAGCTGTAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAAAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGACTGCACAGCGGCGTGCCAAGCAGATTTTCTGGCAGCGGCTCTGGCACCGACTACAGCCTGACAATCAGCAACCTGGAACAAGAGGATATCGCTACCTACTTCTGCCAGCAAGGCAACACCCTGCCTTACACCTTTGGCGGAGGCACCAAGCTGGAAATCACCGGCTCTACAAGCGGCAGCGGCAAACCTGGATCTGGCGAGGGATCTACCAAGGGCGAAGTGAAACTGCAAGAGTCTGGCCCTGGACTGGTGGCCCCATCTCAGTCTCTGAGCGTGACCTGTACAGTCAGCGGAGTGTCCCTGCCTGATTACGGCGTGTCCTGGATCAGACAGCCTCCTCGGAAAGGCCTGGAATGGCTGGGAGTGATCTGGGGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTATTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGATTATTGGGGCCAGGGCACCAGCGTGACCGTTTCTTCTtccggaACCACAACGCCCGCCCCGCGACCGCCTACTCCCGCTCCCACAATTGCATCACAACCCCTGTCTTTGAGACCCGAAGCTTGTCGACCAGCTGCCGGTGGCGCGGTTCACACGCGGGGGCTCGATTTCGCCTGTGATATATATATATGGGCCCCATTGGCTGGAACATGCGGAGTATTGCTTCTGAGCCTGGTGATTACCCTCTACTGTAAGAGAGGCCGGAAGAAACTTCTTTATATATTCAAGCAGCCCTTTATGCGACCCGTTCAGACTACCCAAGAGGAAGATGGATGCAGTTGCCGCTTTCCAGAAGAGGAGGAGGGCGGGTGCGAACTGAGAGTGAAGTTCAGCAGATCCGCCGATGCTCCCGCCTATAAGCAGGGCCAAAACCAGCTGTACAACGAGCTGAACCTGGGGAGAAGAGAAGAGTACGACGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAAATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAGGGCCTGTATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTACCAGGGACTGAGCACCGCCACCAAGGATACCTATGACGCCCTGCAC ATGCAGGCCCTGCCTCCAAGAtaa

In some embodiments, a CAR is encoded by a nucleotide sequence as listedin Table 24.

TABLE 25 CAR domain sequences. SEQ ID NO Protein Sequence 318 4-1BBKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 319 CD3ζRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPE intracellularMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD domainGLYQGLSTATKDTYDALHMQALPPR 320 CD28QVQLVQSGAEVEKPGASVKVSCKASGYTFTDYYMHWVRQAPGQ intracellularGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLR signalingSDDTAVYYCASGWDFDYWGQGTLVTVSSGGGGSGGGGSGGGGS domainGGGGSDIVMTQSPSSLSASVGDRVTITCRASQSIRYYLSWYQQKPGKAPKLLIYTASILQNGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CLQTYTTPDFGPGTKVEIK 321FMC63 VH EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS 322 FMC63 VLDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGN TLPYTFGGGTKLEIT

In some embodiments, a CAR domain encoded by an inventive polynucleotidehas a sequence as listed in Table 25.

TABLE 26 PD-1 or PD-L1 sequences. SEQ ID NO Description Sequence 323Pembrolizumab heavy QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWV chainRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVM HEALHNHYTQKSLSLSLGK 324Pembrolizumab light EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWY chainQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC 325Nivolumab heavy QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVR chainQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH YTQKSLSLSLGK 326Nivolumab light chain EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC 327Atezolizumab heavy EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQ chainAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 328Atezolizumab light DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQK chainPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC 329Avelumab heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 330Avelumab light chain QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQ VTHEGSTVEKTVAPTECS 331Durvalumab heavy EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVR chainQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK 332Durvalumab light EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQK chainPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC

In some embodiments, a cleavage site separating expression sequencesencoded by an inventive polynucleotide has a sequence listed in Table26.

TABLE 27 Cytokine sequences. SEQ ID NO Cytokine Sequence 333 IL-2APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 334 IL-12ARNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIR AVTIDRVMSYLNAS 335IL-12B IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS 336 IL-7DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRIaCDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTIITNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLL QEIKTCWNKILMGTKEH 337 IL-10SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLFFVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQE KGIYKAMSEFDIFINYIEAYMTMKIRN338 IL-15 NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELE EKNIKEFLQSFVHIVQMFINTS339 IL-18 YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKK EDELGDRSIMFTVQNED 340IL-27beta RKGPPAALTLPRVQCRASRYPIAVDCSWTLPPAPNSTSPVSFIATYRLGMAARGHSWPCLQQTPTSTSCTITDVQLFSMAPYVLNVTAVHPWGSSSSFVPFITEHIIKPDPPEGVRLSPLAERQLQVQWEPPGSWPFPEIFSLKYWIRYKRQGAARFHRVGPIEATSFILRAVRPRARYYVQVAAQDLTDYGELSDWSLPATATMSLGK 341 IFNgammaQDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQ MLFRG 342 TGFbeta1ALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIV YYVGRKPKVEQLSNMIVRSCKCS

In some embodiments, a cytokine encoded by an inventive polynucleotidehas a sequence as listed in Table 27.

TABLE 28 Transcription factor sequences. SEQ ID Transcription NO factorSequence 343 FOXP3 MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPGP 344 FOXP3MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFV RVESEKGAVWTVDELEFRKKR 345FOXP3 GGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESE KGAVWTVDELEFRKKR 346 STAT5BMAVWIQAQQLQGEALHQMQALYGQHFPIEVRHYLSQWIESQAWDSVDLDNPQENIKATQLLEGLVQELQKKAEHQVGEDGFLLKIKLGHYATQLQNTYDRCPMELVRCIRHILYNEQRLVREANNGSSPAGSLADAMSQKHLQINQTFEELRLVTQDTENELKKLQQTQEYFTIQYQESLRIQAQFGPLAQLSPQERLSRETALQQKQVSLEAWLQREAQTLQQYRVELAEKHQKTLQLLRKQQTIILDDELIQWKRRQQLAGNGGPPEGSLDVLQSWCEKLAEIIWQNRQQIRRAEHLCQQLPIPGPVEEMLAEVNATITDIISALVTSTFIIEKQPPQVLKTQTKFAATVRLLVGGKLNVHMNPPQVKATIISEQQAKSLLKNENTRNDYSGEILNNCCVMEYHQATGTLSAHFRNMSLKRIKRSDRRGAESVTEEKFTILFESQFSVGGNELVFQVKTLSLPVVVIVHGSQDNNATATVLWDNAFAEPGRVPFAVPDKVLWPQLCEALNMKFKAEVQSNRGLTKENLVFLAQKLFNNSSSHLEDYSGLSVSWSQFNRENLPGRNYTFWQWFDGVMEVLKKHLKPHWNDGAILGFVNKQQAHDLLINKPDGTFLLRFSDSEIGGITIAWKFDSQERMFWNLMPFTTRDFSIRSLADRLGDLNYLIYVFPDRPKDEVYSKYYTPVPCESATAKAVDGYVKPQIKQVVPEFVNASADAGGGSATYMDQAPSPAVCPQAHYNMYPQNPDSVLDTDGDFDLEDTMDVARRVEELLGRPMDSQWIPHAQS 347 HELIOSMETEAIDGYITCDNELSPEREHSNMAIDLTSSTPNGQHASPSHMTSTNSVKLEMQSDEECDRKPLSREDEIRGHDEGSSLEEPLIESSEVADNRKVQELQGEGGIRLPNGKLKCDVCGMVCIGPNVLMVHKRSHTGERPFHCNQCGASFTQKGNLLRHIKLHSGEKPFKCPFCSYACRRRDALTGHLRTHSVGKPHKCNYCGRSYKQRSSLEEHKERCHNYLQNVSMEAAGQVMSHHVPPMEDCKEQEPIMDNNISLVPFERPAVIEKLTGNMGKRKSSTPQKFVGEKLMRFSYPDIHFDMNLTYEKEAELMQSHMMDQAINNAITYLGAEALHPLMQHPPSTIAEVAPVISSAYSQVYHPNRIERPISRETADSHENNMDGPISLIRPKSRPQEREASPSNSCLDSTDSESSHDDHQSYQGHPALNPKRKQSPAYMKEDVKALDTTKAPKGSLKDIYKVFNGEGEQIRAFKCEHCRVLFLDHVMYTIHMGCHGYRDPLECNICGYRSODRYEFSSHIVRGEHTFH

In some embodiments, a transcription factor encoded by an inventivepolynucleotide has a sequence as listed in Table 28.

TABLE 29 Additional Accessory Sequences SEQ ID NO IRES Sequence 390CK 3′ UTR Scrccctgcagccgtcaccgtaagtttgaagttaccgcatatcagcctctgcttcccagcgcgtccaattcctgttcttattgtttcccctccaggcgttacgcgtgacgacgaactgtgtcgcagctaccacattattccggagccttcattctcgcggctctgatcgt 391 CK 3′ UTR S2Mggagaccgcggccacgccgagtaggatcgagggtacagtctcc 392 CK 3′ UTRgacaccaggatcactcttgctctgacccgccctgtgtagaatagactcatgcttccctaagacctggatttcttcccaggcactttcacccgcctgccctgctccttcagtggactgcacccagggaggcggtctctgactgtcctttactttctattctggattgc 393 CK 5′ UTR 1AAACCCCCCTAAGCCGCCGCCGCCGCCACC 394 CK 5′ UTR 2 CCCCCCCAACCCGTCACG 395CK 5′ UTR 3 GTCACG 396 SZ1 3′ UTR Scrtctgcgcactcgtaatcagtactaacccccctttgtcggacactatgcgataatcgatccgcctttttcaccgccttcggaattttatttacctcaactgatcctggagtctctcttggttttcacggaggcctccgccca 397 SZ1 S2M ggagaccgcggccacgccgagtaggatcgagggtacagtctcc 398SZ1 3′ UTRccccttgaaacccccgccccaggttcagtctctcttcatccctctgtcctgcatggtgatacaaagaccctttgtggaccctaagccatgtagttgctgctccctccttccagttgtgaatattggtttctgttaatcaca 399 SZ1 5′ UTR 1 AAACCCCCCTAAGCCGCCGCCGCCGCCACC 400 SZ1 5′ UTR 2CCCCCCCAACCCGTCACG 401 SZ1 5′ UTR 3 GTCACG 402 UTR1 gTcacG 403 UTR2AATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGC CACC 404 UTR3cgaactagtattcttctggtccccacagactcagagagaacccgccacc 405 UTR4 Agccacc 406STOP1 tgatAGctAaCtaG 407 STOP2 tagtAGctAaCtaG 408 STOP3 tGatGActGaGtGA409 STOP4 tagtagctagGtag 410 STOP5 taa 411 STOP6 taatagCtaaCtag 412STOP7 taaCtagCtaaCtag

In some embodiments, a circular RNA or a precursor RNA (e.g., linearprecursor RNA) disclosed herein comprises a sequence as listed in Table29.

In some embodiments, a polynucleotide or a protein encoded by apolynucleotide contains a sequence with at least about 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%similarity to one or more sequences disclosed herein. In someembodiments, a polynucleotide or a protein encoded by a polynucleotidecontains a sequence that is identical to one or more sequences disclosedherein.

Preferred embodiments are described herein. Variations of thosepreferred embodiments may become apparent to those of ordinary skill inthe art upon reading the foregoing description. The inventors expectskilled artisans to employ such variations as appropriate, and theinventors intend for the invention to be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicatedherein or otherwise clearly contradicted by context.

EXAMPLES

Wesselhoeft et al. (2019) RNA Circularization Diminishes Immunogenicityand Can Extend Translation Duration In Vivo. Molecular Cell. 74(3),508-520 and Wesselhoeft et al. (2018) Engineering circular RNA forPotent and Stable Translation in Eukaryotic Cells. NatureCommunications. 9, 2629 are incorporated by reference in their entirety.

The invention is further described in detail by reference to thefollowing examples but are not intended to be limited to the followingexamples. These examples encompass any and all variations of theillustrations with the intention of providing those of ordinary skill inthe art with complete disclosure and description of how to make and usethe subject invention and are not intended to limit the scope of what isregarded as the invention.

Example 1 Example 1A: External Homology Regions Allow forCircularization of Long Precursor RNA Using the Permuted Intron Exon(PIE) Circularization Strategy

A 1,100 nt sequence containing a full-length encephalomyocarditis virus(EMCV) IRES, a Gaussia luciferase (GLuc) expression sequence, and twoshort exon fragments of the permuted intron-exon (PIE) construct wereinserted between the 3′ and 5′ introns of the permuted group I catalyticintron in the thymidylate synthase (Td) gene of the T4 phage. PrecursorRNA was synthesized by run-off transcription. Circularization wasattempted by heating the precursor RNA in the presence of magnesium ionsand GTP, but splicing products were not obtained.

Perfectly complementary 9 nucleotide and 19 nucleotide long homologyregions were designed and added at the 5′ and 3′ ends of the precursorRNA. Addition of these homology arms increased splicing efficiency from0 to 16% for 9 nucleotide homology regions and to 48% for 19 nucleotidehomology regions as assessed by disappearance of the precursor RNA band.

The splicing product was treated with RNase R. Sequencing across theputative splice junction of RNase R-treated splicing reactions revealedligated exons, and digestion of the RNase R-treated splicing reactionwith oligonucleotide-targeted RNase H produced a single band in contrastto two bands yielded by RNase H-digested linear precursor. This showsthat circular RNA is a major product of the splicing reactions ofprecursor RNA containing the 9 or 19 nucleotide long external homologyregions.

Example 1B: Spacers that Conserve Secondary Structures of IRES and PIESplice Sites Increase Circularization Efficiency

A series of spacers was designed and inserted between the 3′ PIE splicesite and the IRES. These spacers were designed to either conserve ordisrupt secondary structures within intron sequences in the IRES, 3′ PIEsplice site, and/or 5′ splice site. The addition of spacer sequencesdesigned to conserve secondary structures resulted in 87% splicingefficiency, while the addition of a disruptive spacer sequences resultedin no detectable splicing.

Example 2 Example 2A: Internal Homology Regions in Addition to ExternalHomology Regions Creates a Splicing Bubble and Allows for Translation ofSeveral Expression Sequences

Spacers were designed to be unstructured, non-homologous to the intronand IRES sequences, and to contain spacer-spacer homology regions. Thesewere inserted between the 5′ exon and IRES and between the 3′ exon andexpression sequence in constructs containing external homology regions,EMCV IRES, and expression sequences for Gaussia luciferase (totallength: 1289 nt), Firefly luciferase (2384 nt), eGFP (145 int), humanerythropoietin (1313 nt), and Cas9 endonuclease (4934 nt).Circularization of all 5 constructs was achieved. Circularization ofconstructs utilizing T4 phage and Anabaena introns were roughly equal.Circularization efficiency was higher for shorter sequences. To measuretranslation, each construct was transfected into HEK293 cells. Gaussiaand Firefly luciferase transfected cells produced a robust response asmeasured by luminescence, human erythropoietin was detectable in themedia of cells transfected with erythropoietin circRNA, and EGFPfluorescence was observed from cells transfected with EGFP circRNA.Co-transfection of Cas9 circRNA with sgRNA directed against GFP intocells constitutively expressing GFP resulted in ablated fluorescence inup to 97% of cells in comparison to an sgRNA-only control.

Example 2B: Use of CVB3 IRES Increases Protein Production

Constructs with internal and external homology regions and differingIRES containing either Gaussia luciferase or Firefly luciferaseexpression sequences were made. Protein production was measured byluminescence in the supernatant of HEK293 cells 24 hours aftertransfection. The Coxsackievirus B3 (CVB3) IRES construct produced themost protein in both cases.

Example 2C: Use of polyA or polyAC Spacers Increases Protein Production

Thirty nucleotide long polyA or polyAC spacers were added between theIRES and splice junction in a construct with each IRES that producedprotein in example 2B. Gaussia luciferase activity was measured byluminescence in the supernatant of HEK293 cells 24 hours aftertransfection. Both spacers improved expression in every construct overcontrol constructs without spacers.

Example 3

HEK293 or HeLa Cells Transfected with Circular RNA Produce More Proteinthan Those Transfected with Comparable Unmodified or Modified LinearRNA.

HPLC-purified Gaussia luciferase-coding circRNA (CVB3-GLuc-pAC) wascompared with a canonical unmodified 5′ methylguanosine-capped and 3′polyA-tailed linear GLuc mRNA, and a commercially availablenucleoside-modified (pseudouridine, 5-methylcytosine) linear GLuc mRNA(from Trilink). Luminescence was measured 24 h post-transfection,revealing that circRNA produced 811.2% more protein than the unmodifiedlinear mRNA in HEK293 cells and 54.5% more protein than the modifiedmRNA Similar results were obtained in HeLa cells and a comparison ofoptimized circRNA coding for human erythropoietin with linear mRNAmodified with 5-methoxyuridine.

Luminescence data was collected over 6 days. In HEK293 cells, circRNAtransfection resulted in a protein production half-life of 80 hours, incomparison with the 43 hours of unmodified linear mRNA and 45 hours ofmodified linear mRNA. In HeLa cells, circRNA transfection resulted in aprotein production half-life of 116 hours, in comparison with the 44hours of unmodified linear mRNA and 49 hours of modified linear mRNA.CircRNA produced substantially more protein than both the unmodified andmodified linear mRNAs over its lifetime in both cell types.

Example 4 Example 4A: Purification of circRNA by RNase Digestion, HPLCPurification, and Phosphatase Treatment Decreases Immunogenicity.Completely Purified Circular RNA is Significantly Less Immunogenic thanUnpurified or Partially Purified Circular RNA. Protein ExpressionStability and Cell Viability are Dependent on Cell Type and Circular RNAPurity

Human embryonic kidney 293 (HEK293) and human lung carcinoma A549 cellswere transfected with:

-   -   products of an unpurified GLuc circular RNA splicing reaction,    -   products of RNase R digestion of the splicing reaction,    -   products of RNase R digestion and HPLC purification of the        splicing reaction, or    -   products of RNase digestion, HPLC purification, and phosphatase        treatment of the splicing reaction.

RNase R digestion of splicing reactions was insufficient to preventcytokine release in A549 cells in comparison to untransfected controls.

The addition of HPLC purification was also insufficient to preventcytokine release, although there was a significant reduction ininterleukin-6 (IL-6) and a significant increase in interferon-α1(IFN-α1) compared to the unpurified splicing reaction.

The addition of a phosphatase treatment after HPLC purification andbefore RNase R digestion dramatically reduced the expression of allupregulated cytokines assessed in A549 cells. Secreted monocytechemoattractant protein 1 (MCP1), IL-6, IFN-α1, tumor necrosis factor α(TNFα), and IFNγ inducible protein-10 (IP-10) fell to undetectable oruntransfected baseline levels.

There was no substantial cytokine release in HEK293 cells. A549 cellshad increased GLuc expression stability and cell viability whentransfected with higher purity circular RNA. Completely purifiedcircular RNA had a stability phenotype similar to that of transfected293 cells.

Example 4B: Circular RNA does not Cause Significant Immunogenicity andis not a RIG-I Ligand

A549 cells were transfected with the products of a splicing reaction:

A549 cells were transfected with:

-   -   unpurified circular RNA,    -   high molecular weight (linear and circular concatenations) RNA,    -   circular (nicked) RNA,    -   an early fraction of purified circular RNA (more overlap with        nicked RNA peak),    -   a late fraction of purified circular RNA (less overlap with        nicked RNA peak),    -   introns excised during circularization, or    -   vehicle (i.e. untransfected control).

Precursor RNA was separately synthesized and purified in the form of thesplice site deletion mutant (DS) due to difficulties in obtainingsuitably pure linear precursor RNA from the splicing reaction. Cytokinerelease and cell viability was measured in each case.

Robust IL-6, RANTES, and IP-10 release was observed in response to mostof the species present within the splicing reaction, as well asprecursor RNA. Early circRNA fractions elicited cytokine responsescomparable to other non-circRNA fractions, indicating that evenrelatively small quantities of linear RNA contaminants are able toinduce a substantial cellular immune response in A549 cells. LatecircRNA fractions elicited no cytokine response in excess of that fromuntransfected controls. A549 cell viability 36 hours post-transfectionwas significantly greater for late circRNA fractions compared with allof the other fractions.

RIG-I and IFN-β1 transcript induction upon transfection of A549 cellswith late circRNA HPLC fractions, precursor RNA or unpurified splicingreactions were analyzed. Induction of both RIG-I and IFN-β1 transcriptswere weaker for late circRNA fractions than precursor RNA and unpurifiedsplicing reactions. RNase R treatment of splicing reactions alone wasnot sufficient to ablate this effect. Addition of very small quantitiesof the RIG-I ligand 3p-hpRNA to circular RNA induced substantial RIG-Itranscription. In HeLa cells, transfection of RNase R-digested splicingreactions induced RIG-I and IFN-β1, but purified circRNA did not.Overall, HeLa cells were less sensitive to contaminating RNA speciesthan A549 cells.

A time course experiment monitoring RIG-I, IFN-β1, IL-6, and RANTEStranscript induction within the first 8 hours after transfection of A549cells with splicing reactions or fully purified circRNA did not reveal atransient response to circRNA. Purified circRNA similarly failed toinduce pro-inflammatory transcripts in RAW264.7 murine macrophages.

A549 cells were transfected with purified circRNA containing an EMCVIRES and EGFP expression sequence. This failed to produce substantialinduction of pro-inflammatory transcripts. These data demonstrate thatnon-circular components of the splicing reaction are responsible for theimmunogenicity observed in previous studies and that circRNA is not anatural ligand for RIG-I.

Example 5

Circular RNA Avoids Detection by TLRs.

TLR 3, 7, and 8 reporter cell lines were transfected with multiplelinear or circular RNA constructs and secreted embryonic alkalinephosphatase (SEAP) was measured.

Linearized RNA was constructed by deleting the intron and homology armsequences. The linear RNA constructs were then treated with phosphatase(in the case of capped RNAs, after capping) and purified by HPLC.

None of the attempted transfections produced a response in TLR7 reportercells. TLR3 and TLR8 reporter cells were activated by capped linearizedRNA, polyadenylated linearized RNA, the nicked circRNA HPLC fraction,and the early circRNA fraction. The late circRNA fraction and m1ψ-mRNAdid not provoke TLR-mediated response in any cell line.

In a second experiment, circRNA was linearized using two methods:treatment of circRNA with heat in the presence of magnesium ions and DNAoligonucleotide-guided RNase H digestion. Both methods yielded amajority of full-length linear RNA with small amounts of intact circRNA.TLR3, 7, and 8 reporter cells were transfected with circular RNA,circular RNA degraded by heat, or circular RNA degraded by RNase H, andSEAP secretion was measured 36 hours after transfection. TLR8 reportercells secreted SEAP in response to both forms of degraded circular RNA,but did not produce a greater response to circular RNA transfection thanmock transfection. No activation was observed in TLR3 and TLR7 reportercells for degraded or intact conditions, despite the activation of TLR3by in vitro transcribed linearized RNA.

Example 6

Unmodified Circular RNA Produces Increased Sustained In Vivo ProteinExpression than Linear RNA.

Mice were injected and HEK293 cells were transfected with unmodified andm1ψ-modified human erythropoietin (hEpo) linear mRNAs and circRNAs.Equimolar transfection of m1ψ-mRNA and unmodified circRNA resulted inrobust protein expression in HEK293 cells. hEpo linear mRNA and circRNAdisplayed similar relative protein expression patterns and cellviabilities in comparison to GLuc linear mRNA and circRNA upon equalweight transfection of HEK293 and A549 cells.

In mice, hEpo was detected in serum after the injection of hEpo circRNAor linear mRNA into visceral adipose. hEpo detected after the injectionof unmodified circRNA decayed more slowly than that from unmodified orm1ψ-mRNA and was still present 42 hours post-injection. Serum hEporapidly declined upon the injection of unpurified circRNA splicingreactions or unmodified linear mRNA. Injection of unpurified splicingreactions produced a cytokine response detectable in serum that was notobserved for the other RNAs, including purified circRNA.

Example 7

Circular RNA can be Effectively Delivered In Vivo or In Vitro Via LipidNanoparticles.

Purified circular RNA was formulated into lipid nanoparticles (LNPs)with the ionizable lipidoid cKK-E12 (Dong et al., 2014; Kauffman et al.,2015). The particles formed uniform multilamellar structures with anaverage size, polydispersity index, and encapsulation efficiency similarto that of particles containing commercially available control linearmRNA modified with 5 moU.

Purified hEpo circRNA displayed greater expression than 5 moU-mRNA whenencapsulated in LNPs and added to HEK293 cells. Expression stabilityfrom LNP-RNA in HEK293 cells was similar to that of RNA delivered bytransfection reagent, with the exception of a slight delay in decay forboth 5 moU-mRNA and circRNA. Both unmodified circRNA and 5 moU-mRNAfailed to activate RIG-I/IFN-β1 in vitro.

In mice, LNP-RNA was delivered by local injection into visceral adiposetissue or intravenous delivery to the liver. Serum hEpo expression fromcircRNA was lower but comparable with that from 5 moU-mRNA 6 hours afterdelivery in both cases. Serum hEpo detected after adipose injection ofunmodified LNP-circRNA decayed more slowly than that from LNP-5moU-mRNA, with a delay in expression decay present in serum that wassimilar to that noted in vitro, but serum hEpo after intravenousinjection of LNP-circRNA or LNP-5 moU-mRNA decayed at approximately thesame rate. There was no increase in serum cytokines or local RIG-I,TNFα, or IL-6 transcript induction in any of these cases.

Example 8 Example 8A: Expression and Functional Stability by IRES inHEK293, HepG2, and 1C1C7 Cells

Constructs including Anabaena intron/exon regions, a Gaussia luciferaseexpression sequence, and varying IRES were circularized. 100 ng of eachcircularization reaction was separately transfected into 20,000 HEK293cells, HepG2 cells, and 1C1C7 cells using Lipofectamine MessengerMax.Luminescence in each supernatant was assessed after 24 hours as ameasure of protein expression. In HEK293 cells, constructs includingCrohivirus B, Salivirus FHB, Aichi Virus, Salivirus HG-J1, andEnterovirus J IRES produced the most luminescence at 24 hours (FIG. 1A).In HepG2 cells, constructs including Aichi Virus, Salivirus FHB,EMCV-Cf, and CVA3 IRES produced high luminescence at 24 hours (FIG. 1B).In 1C1C7 cells, constructs including Salivirus FHB, Aichi Virus,Salivirus NG-J1, and Salivirus A SZ-1 IRES produced high luminescence at24 hours (FIG. 1C).

A trend of larger IRES producing greater luminescence at 24 hours wasobserved. Shorter total sequence length tends to increasecircularization efficiency, so selecting a high expression andrelatively short IRES may result in an improved construct. In HEK293cells, a construct using the Crohivirus B IRES produced the highestluminescence, especially in comparison to other IRES of similar length(FIG. 2A). Expression from IRES constructs in HepG2 and 1C1C7 cellsplotted against IRES size are in FIGS. 2B and 2C.

Functional stability of select IRES constructs in HepG2 and 1C1C7 cellswere measured over 3 days. Luminescence from secreted Gaussia luciferasein supernatant was measured every 24 hours after transfection of 20,000cells with 100 ng of each circularization reaction, followed by completemedia replacement. Salivirus A GUT and Salivirus FHB exhibited thehighest functional stability in HepG2 cells, and Salivirus N-J1 andSalivirus FHB produced the most stable expression in 1C1C7 cells (FIGS.3A and 3B).

Example 8B: Screening of Additional IRES

Functional stability of additional IRES constructs in HEK293 cells weremeasured. Briefly, 5′ untranslated regions (UTRs) of interest wereidentified from GenBank. Selected UTRs were truncated to 675 nt from the5′ end and inserted into a circular RNA backbone construct encodingGaussia Luciferase (Gluc) and in front of the Gluc coding region. Thecircular RNAs were transfected into HEK293 cells. After 24 hours, thesupernatants were collected and the luminescence from secreted Glucprotein was measured using commercially available reagents. The resultsare depicted in FIGS. 1D and 1E and Table 30, suggesting that manynatural IRES sequences enhance the protein expression in a circular RNAcontext.

TABLE 30 SEQ ID NO IRES Expression 413 RhPV 1.10E+05 414 Halastavi arva(1x mut) 9.46E+04 415 Oscivirus 4.55E+07 416 Cadicivirus B 2.10E+05 417PSIV (2x mut for 9.70E+04 Xba1) 418 PSIV IGR 1.01E+05 419 PV Mahoney1.09E+05 420 REV A 9.44E+04 421 Tropivirus A 9.52E+04 422 Symapivirus A1.27E+05 423 Sakobuvirus A FFUP1 8.82E+06 (1x mut) 424 Rosavirus CNFSM6F 6.84E+05 425 Rosavirus 2 GA7403 5.05E+06 426 Rhimavirus A8.42E+05 427 Rafivirus 2.22E+05 LPXYC222841 428 Rafivirus 4.53E+06WHWGGF74766 429 Poecivirus BCCH-449 3.43E+05 430 Megirivirus A LY1.80E+06 431 Megirivirus E 1.10E+07 432 Megirivirus C 1.24E+05 433Ludopivirus 1.05E+05 434 Livupivirus 2.10E+05 435 Aichivirus A FSS6936.25E+07 436 Aichivirus KVGH 1.72E+07 437 Aichivirus DV 7.79E+07 438Murine Kobuvirus 1 1.60E+07 439 Porcine Kobuvirus K- N/A 30 440 PorcineKobuvirus XX 1.32E+07 441 Caprine Kobuvirus 2.87E+08 12Q108 442 RabbitKobuvirus 3.73E+07 443 Aalivirus 2.65E+05 444 Grusopivirus A 1.09E+05445 Grusopivirus B 2.12E+05 446 Yanchengosbecks 1.57E+06 grenadieranchovy picornavirus 447 Turkey Gallivirus 4.37E+05 M176 448 FalcovirusA1 1.48E+05 449 Tremovirus B 1.31E+05 450 Didelphis aurita HAV 1.38E+05451 Hepatovirus G1 1.41E+05 452 Hepatovirus D 1.47E+06 453 HepatovirusH2 1.08E+05 454 Hepatovirus I 8.79E+05 455 Hepatovirus C 5.08E+05 456Fipivirus A 2.69E+05 457 Fipivirus C 1.09E+05 458 Fipivirus E 1.10E+05459 Aquamavirus 4.51E+06 460 Avisivirus A 1.91E+05 461 Avisivirus B8.68E+04 462 Crohivirus A 9.96E+04 463 Kunsagivirus B 8.01E+04 464Limnipivirus A 8.30E+04 465 Limnipivirus C 1.35E+05 466 Orivirus6.09E+05 467 HAV FH1 1.24E+05 468 HAV HM175 4.96E+05 469 Parechovirus F6.56E+05 470 Parechovirus D 3.10E+05 471 Parechovirus C 1.24E+06 472Ljungan Virus 87-012 2.00E+06 473 Parechovirus A2 1.80E+07 474Parechovirus A3 3.58E+06 475 Parechovirus A8 1.61E+07 476 ParechovirusA17 1.20E+06 477 Potamipivirus A 8.43E+05 478 Potamipivirus B 7.20E+05479 Beihai Conger 1.15E+06 Picornavirus 480 Porcine Sapelovirus N/AJD2011 481 Porcine Sapelovirus 4.34E+06 A2 482 Simian Sapelovirus 16.55E+07 483 Simian Sapelovirus 2 4.24E+07 484 Rabovirus C 2.49E+06 485Rabovirus A NYC-B10 1.24E+06 486 Parabovirus C 1.83E+07 487 ParabovirusB 7.85E+06 488 Parabovirus A3 2.44E+08 489 Felipivirus 127F 8.92E+06 490Boosepivirus A 7.07E+07 491 Boosepivirus B 1.17E+08 492 PhacovirusPf-CHK1 5.87E+06 493 HRVC3 QPM 1.64E+07 494 HRVB27 2.04E+08 495 HRVA731.08E+08 496 EV L 6.49E+07 497 EV K 7.52E+07 498 EV J 1631 9.88E+07 499EVJ N125 2.90E+07 500 EV I 1.31E+08 501 EV F1 BEV 261 1.12E+07 502 EVD94 9.25E+07 503 PV3 1.25E+08 504 EV C102 8.85E+07 505 EV 30 5.48E+06506 SAS 1.61E+08 507 EV A114 1.50E+08 508 Mobovirus A 3.44E+06 509Burpengary Virus 1.09E+07 510 Hunnivirus A1 1.61E+06 511 Hunnivirus A26.38E+06 512 Ia Io 1.35E+06 513 Taura Syndrome Virus 8.30E+05 514 ABPV6.48E+05 515 BRAV-2 3.98E+06 516 BRBV-1 3.34E+06 517 ERAV-1 U188 N/A 518GFTV 1.23E+06 519 SAFV V13C 9.32E+07 520 SAV P-113 4.37E+07 521 VHEV1.74E+08 522 TRV NGS910 3.84E+07 523 EMCV2 RD1338 1.97E+06 524 EMCV1JZ1203 N/A 525 EMCV1 AnrB-3741 2.55E+06 526 Cosavirus D1 2.11E+06 527Cosavirus B1 1.91E+06 528 Cosavirus A SH1 2.16E+06 529 Malagasivirus B5.05E+06 530 Mosavirus A2 SZAL6 8.27E+06 531 SVV 1.06E+06 532 PTV A7.29E+05 533 PTV B 6.02E+06 534 Tottorivirus 2.76E+07 535 Posavirus 11.55E+06 536 A105-675 2.18E+07 537 A110-675 1.24E+08 538 18-675 6.04E+07539 A115-675 5.93E+07 540 A73-675 1.30E+08 541 Kobuvirus 16317 2.03E+07542 Aichivirus Chshc7 1.87E+07 543 Aichivirus Goiania 1.66E+07 544Aichivirus ETHP4 1.78E+07 545 Aichivirus DVI2169 2.98E+06 546 AichivirusDVI2321 6.63E+07 547 Aichivirus rat08 3.51E+07 548 Aichivirus Rt3865.71E+07 549 Norway Rat Pestivirus N/A 550 Porcine Kobuvirus 44200000GS2 551 Kobuvirus SZAL6 98850000 552 Kobuvirus sheep TB3 N/A 553Pronghorn antelope 1.35E+06 pestivirus 554 Porcine pestivirus isolateBungowannah 1.10E+07 555 Porcine pestivirus 1 9.46E+04 556 Pestivirusgiraffe-1 4.72E+05 557 Classical swine fever 3.16E+05 virus 558 Humanpegivirus 6.85E+05 isolate JD2B1I 559 Human pegivirus N/A isolateGBV-C-ZJ 560 Human pegivirus 5.36E+05 isolate JD2B8C 561 Hepatitis GBvirus A N/A 562 Simian pegivirus 8.56E+04 563 Pegivirus I 8.02E+04 564Pegivirus K 8.07E+04 565 Theiler's disease- 7.84E+04 associated virus566 Rodent pegivirus 1.79E+05 567 Human pegivirus 2 3.14E+05 568 GBvirus C/Hepatitis G 1.36E+05 virus 569 Equine Pegivirus 1 8.80E+04 570Culex theileri 8.52E+04 flavivirus 571 Bussuquara virus 8.20E+04 572Zika Virus 8.61E+04 573 Yokose virus 8.55E+04 574 Wesselsbron virus N/A575 Equine hepacivirus 8.40E+04 576 Hepacivirus B 8.84E+04 577Hepacivirus I 7.50E+04 578 Hepacivirus J 7.65E+04 579 Hepacivirus K8.91E+04 580 Icavirus 4.41E+06 581 Antarctic penguin virus 8.42E+04 A582 Forest pouched giant N/A rat arterivirus 583 Avisivirus Pf-CHK11.19E+05 584 Avian paramyxovirus 9.91E+04 penguin 585 Newcastle disease8.86E+04 virus 586 Bat Hp- 8.47E+04 betacoronavilus 587 Basella alba7.65E+04 endornavirus 588 Ball python nidovirus 8.25E+04 589 Batsapelovirus 8.05E+04 590 Bat Picornavirus 3 N/A 591 Bat Picornavirus 27.99E+07 592 Bat Picornavirus 1 1.85E+07 593 Bat Iflavirus 9.76E+04 594Bat dicibavirus 7.43E+04 595 Betacoronavirus 8.96E+04 HKU24 596Betacoronavirus 8.74E+04 England 1 597 Boone cardiovirus 1 2.62E+06 598Breda virus 1.16E+05 599 Bovine viral diarrhea 2.70E+06 virus 3 600Bovine rhinitis A virus 3.62E+06 601 Bovine picornavirus 1.21E+05isolate TCH6 602 Bovine nidovirus 1.17E+05 TCH5 603 Bovine hepacivirus1.89E+05 604 Botrytis cinerea 9.68E+04 mitovirus 4 RdRp 605 Botrytiscinerea 8.73E+04 mitovirus 2 RdRp 606 Canine 2.79E+06 picodicistrovirusstrain 209 607 Canine distemper virus 3.02E+05 608 Canine kobuvirus1.48E+08 609 Camel 2.48E+05 alphacoronavirus 610 Cripavirus 1.95E+05 611Human coxsackievirus 7.75E+07 A2 612 Coronavirus AcCoV- 1.82E+05 JC34613 Chicken picornavirus 3 9.13E+04 614 Chicken picornavirus 1 1.21E+05615 Chicken orivirus 1 3.16E+05 616 Chicken gallivirus 1 1.51E+07 617Chicken calicivirus 1.28E+05 618 Carp picornavirus 1 1.13E+05 619 Falconpicornavirus 3.08E+06 620 Equine rhinitis B virus 1.01E+05 1 621 Equinerhinitis A virus 3.73E+05 622 Equine arteritis virus 1.89E+05 623Enterovirus sp. isolate 6.83E+07 CPML 624 Enterovirus AN12 3.87E+06 625Dolphin morbillivirus 1.22E+05 626 Dianke virus 1.35E+05 627 Guerezahepacivirus 1.38E+05 628 Grapevine associated 1.30E+05 narnavirus-1 629Goat torovirus 1.19E+05 630 Foot-and-mouth 1.12E+05 disease virus Oisolate 631 Feline infectious 1.35E+05 peritonitis virus 632 Farmingtonvirus 1.22E+05 633 Avian infectious 2.84E+05 bronchitis virus 634 Humanrhinovirus 1 7.40E+07 635 EV22 1.95E+07 636 Human TMEV-like 4.48E+07cardiovirus 637 Human coronavilus N/A 229E 638 Hubei zhaovirus-like1.03E+05 virus 1 639 Hubei tombus-like 9.28E+04 virus 9 640 Hubeitombus-like 9.23E+04 virus 32 641 Hubei sobemo-like 1.17E+05 virus 3 642Hubei picorna-like 1.95E+05 virus 2 643 Hepacivirus P 6.04E+05 644Harrier picornavirus 1 1.47E+05 645 Kunsagivirus 1 4.15E+05 646Kagoshima-2-24-KoV 9.30E+07 647 Kashmir bee virus 1.65E+05 648 Jingmenpicorna-like 9.32E+04 virus 649 Mumps virus 1.47E+05 650 Mouse Mosavirus9.00E+04 651 Miniopterus 6.05E+06 schreibersii picornavirus 1 652 Lindavirus 7.37E+05 653 Lesavirus 2 3.67E+07 654 Lesavirus 1 6.37E+06 655Phopivirus strain 1.06E+05 NewEngland 656 Pestivirus strain Aydin3.11E+06 657 Quail picornavirus 6.55E+07 QPV1 658 Porcine sapelovirus 1N/A 659 Porcine reproductive 1.29E+05 and respiratory syndrome virus 2660 Porcine enterovirus 9 3.20E+07 661 Pigeon picornavirus B 1.24E+05662 Picornavirus HK21 4.09E+05 663 Picornavirales Tottori- 9.54E+04 HG1664 Rodent hepatovirus 1.39E+05 665 Rinderpest virus 4.26E+05 666Rabovirus A 2.88E+06 667 Shingleback nidovirus 2.62E+05 1 668 Senecavalley virus 1.46E+07 669 Sclerotinia 1.69E+05 sclerotiorum dsRNAmycovirus-L 670 Yak enterovirus 6.19E+06 671 Wobbly possum 2.60E+05disease virus 672 Avian orthoreovirus 4.37E+05 segment S1 673 CaprineKobuvirus d10 2.20E+08 674 Caprine Kobuvirus d20 2.00E+08 675 CaprineKobuvirus d30 1.87E+08 676 Caprine Kobuvirus d40 2.15E+08 677 CaprineKobuvirus d50 9.65E+07 678 Picornavirales sp. 2'26E+08 isolateRtMruf-PicoV 679 Apodemus agrarius 1.90E+08 picornavirus strainLongquan-Aa118 680 Niviventer confucianus 6.10E+07 picornavirus 681 Batpicornavirus 1.13E+06 isolate BtRs-PicoV 682 Rhinolophus N/Apicornavirus strain Guizhou -Rr100 683 Rhinolophus 3.85E+05 picornavirusstrain Henan-Rf265 684 Human enterovirus 5.49E+05 C105 685 Humanpoliovirus 1 3.94E+05 strain NIE1116623 686 Human enterovirus 1094.92E+05 687 Human poliovirus 2 2.59E+07 strain NIE0811460 688 Bovinepicornavirus 3.82E+06 689 Human poliovirus 1 2.44E+05 strain EQG1419328690 Human poliovirus 2 5.84E+06 isolate IS_061 691 Coxsackievirus B5 N/A692 Coxsackievirus A10 N/A

Example 9

Expression and Functional Stability by IRES in Jurkat Cells.

2 sets of constructs including Anabaena intron/exon regions, a Gaussialuciferase expression sequence, and a subset of previously tested IRESwere circularized. 60,000 Jurkat cells were electroporated with 1 μg ofeach circularization reaction. Luminescence from secreted Gaussialuciferase in supernatant was measured 24 hours after electroporation. ACVB3 IRES construct was included in both sets for comparison betweensets and to previously defined IRES efficacy. CVB1 and Salivirus A SZ1IRES constructs produced the most expression at 24 h. Data can be foundin FIGS. 4A and 4B.

Functional stability of the IRES constructs in each round ofelectroporated Jurkat cells was measured over 3 days. Luminescence fromsecreted Gaussia luciferase in supernatant was measured every 24 hoursafter electroporation of 60,000 cells with 1 μg of each circularizationreaction, followed by complete media replacement (FIGS. 5A and 5B).

Salivirus A SZ1 and Salivirus A BN2 IRES constructs had high functionalstability compared to other constructs.

Example 10

Expression, Functional Stability, and Cytokine Release of Circular andLinear RNA in Jurkat Cells.

A construct including Anabaena intron/exon regions, a Gaussia luciferaseexpression sequence, and a Salivirus FHB IRES was circularized. mRNAincluding a Gaussia luciferase expression sequence and a ˜150 nt polyAtail, and modified to replace 100% of uridine with 5-methoxy uridine(5moU) is commercially available and was purchased from Trilink. 5moUnucleotide modifications have been shown to improve mRNA stability andexpression (Bioconjug Chem. 2016 Mar. 16; 27(3):849-53). Expression ofmodified mRNA, circularization reactions (unpure), and circRNA purifiedby size exclusion HPLC (pure) in Jurkat cells were measured and compared(FIG. 6A). Luminescence from secreted Gaussia luciferase in supernatantwas measured 24 hours after electroporation of 60,000 cells with 1 μg ofeach RNA species.

Luminescence from secreted Gaussia luciferase in supernatant wasmeasured every 24 hours after electroporation of 60,000 cells with 1 ugof each RNA species, followed by complete media replacement. Acomparison of functional stability data of modified mRNA and circRNA inJurkat cells over 3 days is in FIG. 6B.

IFNγ (FIG. 7A), IL-6 (FIG. 7B), IL-2 (FIG. 7C), RIG-I (FIG. 7D), IFN-β1(FIG. 7E), and TNFα (FIG. 7F) transcript induction was measured 18 hoursafter electroporation of 60,000 Jurkat cells with 1 μg of each RNAspecies described above and 3p-hpRNA (5′ triphosphate hairpin RNA, whichis a known RIG-I agonist).

Example 11

Expression of Circular and Linear RNA in Monocytes and Macrophages.

A construct including Anabaena intron/exon regions, a Gaussia luciferaseexpression sequence, and a Salivirus FHB IRES was circularized. mRNAincluding a Gaussia luciferase expression sequence and a ˜150 nt polyAtail, and modified to replace 100% of uridine with 5-methoxy uridine(5moU) was purchased from Trilink. Expression of circular and modifiedmRNA was measured in human primary monocytes (FIG. 8A) and human primarymacrophages (FIG. 8B). Luminescence from secreted Gaussia luciferase insupernatant was measured 24 hours after electroporation of 60,000 cellswith 1 μg of each RNA species. Luminescence was also measured 4 daysafter electroporation of human primary macrophages with media changesevery 24 hours (FIG. 8C). The results can be found in FIG. 8 . Thedifference in luminescence was statistically significant in each case(p<0.05).

Example 12

Expression and Functional Stability by IRES in Primary T Cells.

Constructs including Anabaena intron/exon regions, a Gaussia luciferaseexpression sequence, and a subset of previously tested IRES werecircularized and reaction products were purified by size exclusion HPLC.150,000 primary human CD3+ T cells were electroporated with 1 μg of eachcircRNA. Luminescence from secreted Gaussia luciferase in supernatantwas measured 24 hours after electroporation (FIG. 9A). Aichi Virus andCVB3 IRES constructs had the most expression at 24 hours.

Luminescence was also measured every 24 hours after electroporation for3 days in order to compare functional stability of each construct (FIG.9B). The construct with a Salivirus A SZ1 IRES was the most stable.

Example 13

Expression and Functional Stability of Circular and Linear RNA inPrimary T Cells and PBMCs.

Constructs including Anabaena intron/exon regions, a Gaussia luciferaseexpression sequence, and a Salivirus A SZ1 IRES or Salivirus FHB IRESwere circularized. mRNA including a Gaussia luciferase expressionsequence and a ˜150 nt polyA tail, and modified to replace 100% ofuridine with 5-methoxy uridine (5moU) and was purchased from Trilink.Expression of Salivirus A SZ1 IRES HPLC purified circular and modifiedmRNA was measured in human primary CD3+ T cells. Expression of SalivirusFHB HPLC purified circular, unpurified circular and modified mRNA wasmeasured in human PBMCs. Luminescence from secreted Gaussia luciferasein supernatant was measured 24 hours after electroporation of 150,000cells with 1 μg of each RNA species. Data for primary human T cells isshown in FIGS. 10A and 10B, and data for PBMCs is shown in FIG. 10C. Thedifference in expression between the purified circular RNA andunpurified circular RNA or linear RNA was significant in each case(p<0.05).

Luminescence from secreted Gaussia luciferase in primary T cellsupernatant was measured every 24 hours after electroporation over 3days in order to compare construct functional stability. Data is shownin FIG. 10B. The difference in relative luminescence from the day 1measurement between purified circular RNA and linear RNA was significantat both day 2 and day 3 for primary T cells.

Example 14

Circularization Efficiency by Permutation Site in Anabaena Intron.

RNA constructs including a CVB3 IRES, a Gaussia luciferase expressionsequence, Anabaena intron/exon regions, spacers, internal homologyregions, and homology arms were produced. Circularization efficiency ofconstructs using the traditional Anabaena intron permutation site and 5consecutive permutations sites in P9 was measured by HPLC. HPLCchromatograms for the 5 consecutive permutation sites in P9 are shown inFIG. 11A.

Circularization efficiency was measured at a variety of permutationsites. Circularization efficiency is defined as the area under the HPLCchromatogram curve for each of: circRNA/(circRNA+precursor RNA). Rankedquantification of circularization efficiency at each permutation site isin FIG. 11B. 3 permutation sites (indicated in FIG. 11B) were selectedfor further investigation.

Circular RNA in this example was circularized by in vitro transcription(IVT) then purified via spin column. Circularization efficiency for allconstructs would likely be higher if the additional step of incubationwith Mg2+ and guanosine nucleotide were included; however, removing thisstep allowed for comparison between, and optimization of, circular RNAconstructs. This level of optimization is especially useful formaintaining high circularization efficiency with large RNA constructs,such as those encoding chimeric antigen receptors.

Example 15

Circularization Efficiency of Alternative Introns.

Precursor RNA containing a permuted group 1 intron of variable speciesorigin or permutation site and several constant elements including: aCVB3 IRES, a Gaussia luciferase expression sequence, spacers, internalhomology regions, and homology arms were created. Circularization datacan be found in FIG. 12 . FIG. 12A shows chromatograms resolvingprecursor, CircRNA and introns. FIG. 12B provides ranked quantificationof circularization efficiency, based on the chromatograms shown in FIG.12A, as a function of intron construct.

Circular RNA in this example was circularized by in vitro transcription(IVT) then spin column purification. Circularization efficiency for allconstructs would likely be higher if the additional step of incubationwith Mg2+ and guanosine nucleotide were included; however, removing thisstep allows for comparison between, and optimization of, circular RNAconstructs. This level of optimization is especially useful formaintaining high circularization efficiency with large RNA constructs,such as those encoding chimeric antigen receptors.

Example 16

Circularization Efficiency by Homology Arm Presence or Length.

RNA constructs including a CVB3 IRES, a Gaussia luciferase expressionsequence, Anabaena intron/exon regions, spacers, and internal homologyregions were produced. Constructs representing 3 Anabaena intronpermutation sites were tested with 30 nt, 25% GC homology arms orwithout homology arms (“NA”). These constructs were allowed tocircularize without an Mg²⁺ incubation step. Circularization efficiencywas measured and compared. Data can be found in FIGS. 13A and 13B.Circularization efficiency was higher for each construct lackinghomology arms. FIG. 13A provides ranked quantification ofcircularization efficiency; FIG. 13B provides chromatograms resolvingprecursor, circRNA and introns.

For each of the 3 permutation sites, constructs were created with 10 nt,20 nt, and 30 nt arm lengths and 25%, 50%, and 75% GC content. Splicingefficiency of these constructs was measured and compared to constructswithout homology arms (FIG. 14 ). Splicing efficiency is defined as theproportion of free introns relative to the total RNA in the splicingreaction.

FIG. 15A (left) shows HPLC chromatograms indicating the contribution ofstrong homology arms to improved splicing efficiency. Top left: 75% GCcontent, 10 nt homology arms. Center left: 75% GC content, 20 nthomology arms. Bottom left: 75% GC content, 30 nt homology arms.

FIG. 15A (right) shows HPLC chromatograms showing increased splicingefficiency paired with increased nicking, appearing as a shoulder on thecircRNA peak. Top right: 75% GC content, 10 nt homology arms. Centerright: 75% GC content, 20 nt homology arms. Bottom right: 75% GCcontent, 30 nt homology arms.

FIG. 15B (left) shows select combinations of permutation sites andhomology arms hypothesized to demonstrate improved circularizationefficiency.

FIG. 15B (right) shows select combinations of permutation sites andhomology arms hypothesized to demonstrate improved circularizationefficiency, treated with E. coli polyA polymerase.

Circular RNA in this example was circularized by in vitro transcription(IVT) then spin-column purified. Circularization efficiency for allconstructs would likely be higher if an additional Mg2+ incubation stepwith guanosine nucleotide were included; however, removing this stepallowed for comparison between, and optimization of, circular RNAconstructs. This level of optimization is especially useful formaintaining high circularization efficiency with large RNA constructs,such as those encoding chimeric antigen receptors.

Example 17

Circular RNA Encoding Chimeric Antigen Receptors

Constructs including Anabaena intron/exon regions, a Kymriah chimericantigen receptors (CAR) expression sequence, and a CVB3 IRES werecircularized. 100,000 human primary CD3+ T cells were electroporatedwith 500 ng of circRNA and co-cultured for 24 hours with Raji cellsstably expressing GFP and firefly luciferase. Effector to target ratio(E:T ratio) 0.75:1. 100,000 human primary CD3+ T cells were mockelectroporated and co-cultured as a control (FIG. 16 ).

Sets of 100,000 human primary CD3+ T cells were mock electroporated orelectroporated with 1 μg of circRNA then co-cultured for 48 hours withRaji cells stably expressing GFP and firefly luciferase E:T ratio 10:1(FIG. 17 ).

Quantification of specific lysis of Raji target cells was determined bydetection of firefly luminescence (FIG. 18 ). 100,000 human primary CD3+T cells either mock electroporated or electroporated with circRNAencoding different CAR sequences were co-cultured for 48 hours with Rajicells stably expressing GFP and firefly luciferase. % Specific lysisdefined as 1-[CAR condition luminescence]/[mock condition luminescence].E:T ratio 10:1.

Example 18

Expression and Functional Stability of Circular and Linear RNA in JurkatCells and Resting Human T Cells.

Constructs including Anabaena intron/exon regions, a Gaussia luciferaseexpression sequence, and a subset of previously tested IRES werecircularized and reaction products were purified by size exclusion HPLC.150,000 Jurkat cells were electroporated with 1 μg of circular RNA or5moU-mRNA. Luminescence from secreted Gaussia luciferase in supernatantwas measured 24 hours after electroporation (FIG. 19A left). 150,000resting primary human CD3+ T cells (10 days post-stimulation) wereelectroporated with 1 μg of circular RNA or 5moU-mRNA. Luminescence fromsecreted Gaussia luciferase in supernatant was measured 24 hours afterelectroporation (FIG. 19A right).

Luminescence from secreted Gaussia luciferase in supernatant wasmeasured every 24 hours after electroporation, followed by completemedia replacement. Functional stability data shown in FIG. 19B. CircularRNA had more functional stability than linear RNA in each case, with amore pronounced difference in Jurkat cells.

Example 19

IFN-β1, RIG-I, IL-2, IL-6, IFNγ, and TNFα Transcript Induction of CellsElectroporated with Linear RNA or Varying Circular RNA Constructs.

Constructs including Anabaena intron/exon regions, a Gaussia luciferaseexpression sequence, and a subset of previously tested IRES werecircularized and reaction products were purified by size exclusion HPLC.150,000 CD3+ human T cells were electroporated with 1 μg of circularRNA, 5moU-mRNA, or immunostimulatory positive control polyinosine:cytosine. IFN-β1 (FIG. 20A), RIG-I (FIG. 20B), IL-2 (FIG. 20C),IL-6 (FIG. 20D), IFNγ (FIG. 20E), and TNFα (FIG. 20F) transcriptinduction was measured 18 hours after electroporation.

Example 20

Specific Lysis of Target Cells and IFNγ Transcript Induction by CARExpressing Cells Electroporated with Different Amounts of Circular orLinear RNA; Specific Lysis of Target and Non-Target Cells by CARExpressing Cells at Different E:T Ratios.

Constructs including Anabaena intron/exon regions, an anti-CD19 CARexpression sequence, and a CVB3 IRES were circularized and reactionproducts were purified by size exclusion HPLC. 150,000 human primaryCD3+ T cells either mock electroporated or electroporated with differentquantities of circRNA encoding an anti-CD19 CAR sequence wereco-cultured for 12 hours with Raji cells stably expressing GFP andfirefly luciferase at an E:T ratio of 2:1. Specific lysis of Raji targetcells was determined by detection of firefly luminescence (FIG. 21A). %Specific lysis was defined as 1-[CAR condition luminescence]/[mockcondition luminescence]. IFNγ transcript induction was measured 24 hoursafter electroporation (FIG. 21B).

150,000 human primary CD3+ T cells were either mock electroporated orelectroporated with 500 ng circRNA or m1ψ-mRNA encoding an anti-CD19 CARsequence, then co-cultured for 24 hours with Raji cells stablyexpressing firefly luciferase at different E:T ratios. % Specific lysisof Raji target cells was determined by detection of firefly luminescence(FIG. 22A). % Specific lysis was defined as 1-[CAR conditionluminescence]/[mock condition luminescence].

CAR expressing T cells were also co-cultured for 24 hours with Raji orK562 cells stably expressing firefly luciferase at different E:T ratios.Specific lysis of Raji target cells or K562 non-target cells wasdetermined by detection of firefly luminescence (FIG. 22B). % Specificlysis is defined as 1-[CAR condition luminescence]/[mock conditionluminescence].

Example 21

Specific Lysis of Target Cells by T Cells Electroporated with CircularRNA or Linear RNA Encoding a CAR.

Constructs including Anabaena intron/exon regions, an anti-CD19 CARexpression sequence, and a CVB3 IRES were circularized and reactionproducts were purified by size exclusion HPLC. Human primary CD3+ Tcells were electroporated with 500 ng of circular RNA or an equimolarquantity of m1ψ-mRNA, each encoding a CD19-targeted CAR. Raji cells wereadded to CAR-T cell cultures over 7 days at an E:T ratio of 10:1. %Specific lysis was measured for both constructs at 1, 3, 5, and 7 days(FIG. 23 ).

Example 22

Specific Lysis of Raji Cells by T Cells Expressing an Anti-CD19 CAR oran Anti-BCMA CAR.

Constructs including Anabaena intron/exon regions, anti-CD19 oranti-BCMA CAR expression sequence, and a CVB3 IRES were circularized andreaction products were purified by size exclusion HPLC. 150,000 primaryhuman CD3+ T cells were electroporated with 500 ng of circRNA, then wereco-cultured with Raji cells at an E:T ratio of 2:1. % Specific lysis wasmeasured 12 hours after electroporation (FIG. 24 ).

Example 23 Example 23A: Synthesis of Compounds

Synthesis of representative ionizable lipids of the invention aredescribed in PCT applications PCT/US2016/052352, PCT/US2016/068300,PCT/US2010/061058, PCT/US2018/058555, PCT/US2018/053569,PCT/US2017/028981, PCT/US2019/025246, PCT/US2018/035419,PCT/US2019/015913, and US applications with publication numbers20190314524, 20190321489, and 20190314284, the contents of each of whichare incorporated herein by reference in their entireties.

Example 23B: Synthesis of Compounds

Synthesis of representative ionizable lipids of the invention aredescribed in US patent publication number US20170210697A1, the contentsof which is incorporated herein by reference in its entirety.

Example 24

Protein Expression by Organ

Circular or linear RNA encoding FLuc was generated and loaded intotransfer vehicles with the following formulation: 50% ionizable lipid 15in Table 10b, 10% DSPC, 1.5% PEG-DMG, 38.5% cholesterol. CD-1 mice weredosed at 0.2 mg/kg and luminescence was measured at 6 hours (live IVIS)and 24 hours (live IVIS and ex vivo IVIS). Total Flux (photons/secondover a region of interest) of the liver, spleen, kidney, lung, and heartwas measured (FIGS. 25 and 26 ).

Example 25

Distribution of Expression in the Spleen

Circular or linear RNA encoding GFP is generated and loaded intotransfer vehicles with the following formulation: 50% ionizable lipid 15in Table 10b, 10% DSPC, 1.5% PEG-DMG, 38.5% cholesterol. The formulationis administered to CD-1 mice. Flow cytometry is run on spleen cells todetermine the distribution of expression across cell types.

Example 26

Production of Nanoparticle Compositions

In order to investigate safe and efficacious nanoparticle compositionsfor use in the delivery of circular RNA to cells, a range offormulations are prepared and tested. Specifically, the particularelements and ratios thereof in the lipid component of nanoparticlecompositions are optimized.

Nanoparticles can be made in a 1 fluid stream or with mixing processessuch as microfluidics and T-junction mixing of two fluid streams, one ofwhich contains the circular RNA and the other has the lipid components.

Lipid compositions are prepared by combining an ionizable lipid,optionally a helper lipid (such as DOPE, DSPC, or oleic acid obtainablefrom Avanti Polar Lipids, Alabaster, AL), a PEG lipid (such as1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol, also known asPEG-DMG, obtainable from Avanti Polar Lipids, Alabaster, Ala.), and astructural lipid such as cholesterol at concentrations of about, e.g.,40 or 50 mM in a solvent, e.g., ethanol. Solutions should berefrigerated for storage at, for example, −20° C. Lipids are combined toyield desired molar ratios (see, for example, Tables 31a and 31b below)and diluted with water and ethanol to a final lipid concentration ofe.g., between about 5.5 mM and about 25 mM.

TABLE 31a Formulation number Description 1 Aliquots of 50 mg/mLethanolic solutions of C12-200, DOPE, Chol and DMG- PEG2K (40:30:25:5)are mixed and diluted with ethanol to 3 mL final volume. Separately, anaqueous buffered solution (10 mM citrate/150 mM NaCl, pH 4.5) of circRNAis prepared from a 1 mg/mL stock. The lipid solution is injected rapidlyinto the aqueous circRNA solution and shaken to yield a final suspensionin 20% ethanol. The resulting nanoparticle suspension is filtered,diafiltrated with 1 × PBS (pH 7.4), concentrated and stored at 2-8° C. 2Aliquots of 50 mg/mL ethanolic solutions of DODAP, DOPE, cholesterol andDMG-PEG2K (18:56:20:6) are mixed and diluted with ethanol to 3 mL finalvolume. Separately, an aqueous buffered solution (10mM citrate/150mMNaCl, pH 4.5) of EPO circRNA is prepared from a 1 mg/mL stock. The lipidsolution is injected rapidly into the aqueous circRNA solution andshaken to yield a final suspension in 20% ethanol. The resultingnanoparticle suspension is filtered, diafiltrated with 1 × PBS (pH 7.4),concentrated and stored at 2-8° C. Final concentration = 1.35 mg/mL EPOcircRNA (encapsulated). Zave = 75.9 nm (Dv(50) = 57.3 nm; Dv(90) = 92.1nm). 3 Aliquots of 50 mg/mL ethanolic solutions of HGT4003, DOPE,cholesterol and DMG-PEG2K (50:25:20:5) are mixed and diluted withethanol to 3 mL final volume. Separately, an aqueous buffered solution(10 mM citrate/150 mM NaCl, pH 4.5) of circRNA is prepared from a 1mg/mL stock. The lipid solution is injected rapidly into the aqueouscircRNA solution and shaken to yield a final suspension in 20% ethanol.The resulting nanoparticle suspension is filtered, diafiltrated with 1 ×PBS (pH 7.4), concentrated and stored at 2-8° C. 4 Aliquots of 50 mg/mLethanolic solutions of ICE, DOPE and DMG-PEG2K (70:25:5) are mixed anddiluted with ethanol to 3 mL final volume. Separately, an aqueousbuffered solution (10 mM citrate/150 mM NaCl, pH 4.5) of circRNA isprepared from a 1 mg/mL stock. The lipid solution is injected rapidlyinto the aqueous circRNA solution and shaken to yield a final suspensionin 20% ethanol. The resulting nanoparticle suspension is filtered,diafiltrated with 1 × PBS (pH 7.4), concentrated and stored at 2-8° C. 5Aliquots of 50 mg/mL ethanolic solutions of HGT5000, DOPE, cholesteroland DMG-PEG2K (40:20:35:5) are mixed and diluted with ethanol to 3 mLfinal volume. Separately, an aqueous buffered solution (10 mMcitrate/150 mM NaCl, pH 4.5) of EPO circRNA is prepared from a 1 mg/mLstock. The lipid solution is injected rapidly into the aqueous circRNAsolution and shaken to yield a final suspension in 20% ethanol. Theresulting nanoparticle suspension is filtered, diafiltrated with 1 × PBS(pH 7.4), concentrated and stored at 2-8° C. Final concentration = 1.82mg/mL EPO mRNA (encapsulated). Zave = 105.6 nm (Dv(50) = 53.7 nm; Dv(90)= 157 nm). 6 Aliquots of 50 mg/mL ethanolic solutions of HGT5001, DOPE,cholesterol and DMG-PEG2K (40:20:35:5) are mixed and diluted withethanol to 3 mL final volume. Separately, an aqueous buffered solution(10 mM citrate/150 mM NaCl, pH 4.5) of EPO circRNA is prepared from a 1mg/mL stock. The lipid solution is injected rapidly into the aqueouscircRNA solution and shaken to yield a final suspension in 20% ethanol.The resulting nanoparticle suspension is filtered, diafiltrated with 1 ×PBS (pH 7.4), concentrated and stored at 2-8° C.

In some embodiments, transfer vehicle has a formulation as described inTable 31a.

TABLE 31b Composition (mol %) Components 40:20:38.5:1.5Compound:Phospholipid:Phytosterol*:PEG-DMG 45:15:38.5:1.5Compound:Phospholipid:Phytosterol*:PEG-DMG 50:10:38.5:1.5Compound:Phospholipid:Phytosterol*:PEG-DMG 55:5:38.5:1.5Compound:Phospholipid:Phytosterol*:PEG-DMG 60:5:33.5:1.5Compound:Phospholipid:Phytosterol*:PEG-DMG 45:20:33.5:1.5Compound:Phospholipid:Phytosterol*:PEG-DMG 50:20:28.5:1.5Compound:Phospholipid:Phytosterol*:PEG-DMG 55:20:23.5:1.5Compound:Phospholipid:Phytosterol*:PEG-DMG 60:20:18.5:1.5Compound:Phospholipid:Phytosterol*:PEG-DMG 40:15:43.5:1.5Compound:Phospholipid:Phytosterol*:PEG-DMG 50:15:33.5:1.5Compound:Phospholipid:Phytosterol*:PEG-DMG 55:15:28.5:1.5Compound:Phospholipid:Phytosterol*:PEG-DMG 60:15:23.5:1.5Compound:Phospholipid:Phytosterol*:PEG-DMG 40:10:48.5:1.5Compound:Phospholipid:Phytosterol*:PEG-DMG 45:10:43.5:1.5Compound:Phospholipid:Phytosterol*:PEG-DMG 55:10:33.5:1.5Compound:Phospholipid:Phytosterol*:PEG-DMG 60:10:28.5:1.5Compound:Phospholipid:Phytosterol*:PEG-DMG 40:5:53.5:1.5Compound:Phospholipid:Phytosterol*:PEG-DMG 45:5:48.5:1.5Compound:Phospholipid:Phytosterol*:PEG-DMG 50:5:43.5:1.5Compound:Phospholipid:Phytosterol*:PEG-DMG 40:20:40:0Compound:Phospholipid:Phytosterol*:PEG-DMG 45:20:35:0Compound:Phospholipid:Phytosterol*:PEG-DMG 50:20:30:0Compound:Phospholipid:Phytosterol*:PEG-DMG 55:20:25:0Compound:Phospholipid:Phytosterol*:PEG-DMG 60:20:20:0Compound:Phospholipid:Phytosterol*:PEG-DMG 40:15:45:0Compound:Phospholipid:Phytosterol*:PEG-DMG

In some embodiments, transfer vehicle has a formulation as described inTable 31b.

For nanoparticle compositions including circRNA, solutions of thecircRNA at concentrations of 0.1 mg/ml in deionized water are diluted ina buffer, e.g., 50 mM sodium citrate buffer at a pH between 3 and 4 toform a stock solution. Alternatively, solutions of the circRNA atconcentrations of 0.15 mg/ml in deionized water are diluted in a buffer,e.g., 6.25 mM sodium acetate buffer at a pH between 3 and 4.5 to form astock solution.

Nanoparticle compositions including a circular RNA and a lipid componentare prepared by combining the lipid solution with a solution includingthe circular RNA at lipid component to circRNA wt:wt ratios betweenabout 5:1 and about 50:1. The lipid solution is rapidly injected using,e.g., a NanoAssemblr microfluidic based system at flow rates betweenabout 10 ml/min and about 18 ml/min or between about 5 ml/min and about18 ml/min into the circRNA solution, to produce a suspension with awater to ethanol ratio between about 1:1 and about 4:1.

Nanoparticle compositions can be processed by dialysis to remove ethanoland achieve buffer exchange. Formulations are dialyzed twice againstphosphate buffered saline (PBS), pH 7.4, at volumes 200 times that ofthe primary product using Slide-A-Lyzer cassettes (Thermo FisherScientific Inc., Rockford, IL) with a molecular weight cutoff of 10 kDaor 20 kDa. The formulations are then dialyzed overnight at 4° C. Theresulting nanoparticle suspension is filtered through 0.2 μm sterilefilters (Sarstedt, Nümbrecht, Germany) into glass vials and sealed withcrimp closures. Nanoparticle composition solutions of 0.01 mg/ml to 0.15mg/ml are generally obtained.

The method described above induces nano-precipitation and particleformation.

Alternative processes including, but not limited to, T-junction anddirect injection, may be used to achieve the same nano-precipitation. B.Characterization of nanoparticle compositions

A Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire,UK) can be used to determine the particle size, the polydispersity index(PDI) and the zeta potential of the nanoparticle compositions in 1×PBSin determining particle size and 15 mM PBS in determining zetapotential.

Ultraviolet-visible spectroscopy can be used to determine theconcentration of circRNA in nanoparticle compositions. 100 μL of thediluted formulation in 1×PBS is added to 900 μL of a 4:1 (v/v) mixtureof methanol and chloroform. After mixing, the absorbance spectrum of thesolution is recorded, for example, between 230 nm and 330 nm on a DU 800spectrophotometer (Beckman Coulter, Beckman Coulter, Inc., Brea, CA).The concentration of circRNA in the nanoparticle composition can becalculated based on the extinction coefficient of the circRNA used inthe composition and on the difference between the absorbance at awavelength of, for example, 260 nm and the baseline value at awavelength of, for example, 330 nm.

A QUANT-IT™ RIBOGREEN® RNA assay (Invitrogen Corporation Carlsbad, CA)can be used to evaluate the encapsulation of circRNA by the nanoparticlecomposition. The samples are diluted to a concentration of approximately5 μg/mL or 1 μg/mL in a TE buffer solution (10 mM Tris-HCl, 1 mM EDTA,pH 7.5). 50 μL of the diluted samples are transferred to a polystyrene96 well plate and either 50 μL of TE buffer or 50 μL of a 2-4% TritonX-100 solution is added to the wells. The plate is incubated at atemperature of 37° C. for 15 minutes. The RIBOGREEN® reagent is diluted1:100 or 1:200 in TE buffer, and 100 μL of this solution is added toeach well. The fluorescence intensity can be measured using afluorescence plate reader (Wallac Victor 1420 Multilabel Counter; PerkinElmer, Waltham, MA) at an excitation wavelength of, for example, about480 nm and an emission wavelength of, for example, about 520 nm. Thefluorescence values of the reagent blank are subtracted from that ofeach of the samples and the percentage of free circRNA is determined bydividing the fluorescence intensity of the intact sample (withoutaddition of Triton X-100) by the fluorescence value of the disruptedsample (caused by the addition of Triton X-100). C.

In Vivo Formulation Studies:

In order to monitor how effectively various nanoparticle compositionsdeliver circRNA to targeted cells, different nanoparticle compositionsincluding circRNA are prepared and administered to rodent populations.Mice are intravenously, intramuscularly, intraarterially, orintratumorally administered a single dose including a nanoparticlecomposition with a lipid nanoparticle formulation. In some instances,mice may be made to inhale doses. Dose sizes may range from 0.001 mg/kgto 10 mg/kg, where 10 mg/kg describes a dose including 10 mg of acircRNA in a nanoparticle composition for each 1 kg of body mass of themouse. A control composition including PBS may also be employed.

Upon administration of nanoparticle compositions to mice, dose deliveryprofiles, dose responses, and toxicity of particular formulations anddoses thereof can be measured by enzyme-linked immunosorbent assays(ELISA), bioluminescent imaging, or other methods. Time courses ofprotein expression can also be evaluated. Samples collected from therodents for evaluation may include blood and tissue (for example, muscletissue from the site of an intramuscular injection and internal tissue);sample collection may involve sacrifice of the animals.

Higher levels of protein expression induced by administration of acomposition including a circRNA will be indicative of higher circRNAtranslation and/or nanoparticle composition circRNA deliveryefficiencies. As the non-RNA components are not thought to affecttranslational machineries themselves, a higher level of proteinexpression is likely indicative of a higher efficiency of delivery ofthe circRNA by a given nanoparticle composition relative to othernanoparticle compositions or the absence thereof.

Example 27

Characterization of Nanoparticle Compositions

A Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire,UK) can be used to determine the particle size, the polydispersity index(PDI) and the zeta potential of the transfer vehicle compositions in1×PBS in determining particle size and 15 mM PBS in determining zetapotential.

Ultraviolet-visible spectroscopy can be used to determine theconcentration of a therapeutic and/or prophylactic (e.g., RNA) intransfer vehicle compositions. 100 μL of the diluted formulation in1×PBS is added to 900 μL of a 4:1 (v/v) mixture of methanol andchloroform. After mixing, the absorbance spectrum of the solution isrecorded, for example, between 230 nm and 330 nm on a DU 800spectrophotometer (Beckman Coulter, Beckman Coulter, Inc., Brea, CA).The concentration of therapeutic and/or prophylactic in the transfervehicle composition can be calculated based on the extinctioncoefficient of the therapeutic and/or prophylactic used in thecomposition and on the difference between the absorbance at a wavelengthof, for example, 260 nm and the baseline value at a wavelength of, forexample, 330 nm.

For transfer vehicle compositions including RNA, a QUANT-IT™ RIBOGREEN®RNA assay (Invitrogen Corporation Carlsbad, CA) can be used to evaluatethe encapsulation of RNA by the transfer vehicle composition. Thesamples are diluted to a concentration of approximately 5 μg/mL or 1μg/mL in a TE buffer solution (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). 50 μLof the diluted samples are transferred to a polystyrene 96 well plateand either 50 μL of TE buffer or 50 μL of a 2-4% Triton X-100 solutionis added to the wells. The plate is incubated at a temperature of 37° C.for 15 minutes. The RIBOGREEN® reagent is diluted 1:100 or 1:200 in TEbuffer, and 100 μL of this solution is added to each well. Thefluorescence intensity can be measured using a fluorescence plate reader(Wallac Victor 1420 Multilablel Counter; Perkin Elmer, Waltham, MA) atan excitation wavelength of, for example, about 480 nm and an emissionwavelength of, for example, about 520 nm. The fluorescence values of thereagent blank are

subtracted from that of each of the samples and the percentage of freeRNA is determined by dividing the fluorescence intensity of the intactsample (without addition of Triton X-100) by the fluorescence value ofthe disrupted sample (caused by the addition of Triton X-100).

Example 28

T Cell Targeting

To target transfer vehicles to T-cells, T cell antigen binders, e.g.,anti-CD8 antibodies, are coupled to the surface of the transfer vehicle.Anti-T cell antigen antibodies are mildly reduced with an excess of DTTin the presence of EDTA in PBS to expose free hinge region thiols. Toremove DTT, antibodies are passed through a desalting column. Theheterobifunctional cross-linker SM(PEG)24 is used to anchor antibodiesto the surface of circRNA-loaded transfer vehicles (Amine groups arepresent in the head groups of PEG lipids, free thiol groups onantibodies were created by DTT, SM(PEG)24 cross-links between amines andthiol groups). Transfer vehicles are first incubated with an excess ofSM(PEG)24 and centrifuged to remove unreacted cross-linker. Activatedtransfer vehicles are then incubated with an excess of reduced anti-Tcell antigen antibody. Unbound antibody is removed using a centrifugalfiltration device.

Example 29

RNA Containing Transfer Vehicle Using RV88.

In this example RNA containing transfer vehicles are synthesized usingthe 2-D vortex microfluidic chip with the cationic lipid RV88 fordelivery of circRNA.

TABLE 32a Materials and Instrument Vendor Cat # 1M Tris-HCl, pH 8.0,Sterile Teknova T1080 5M Sodium Chloride solution Teknova S0250 QBCitrate buffer, pH 6.0 (100 mM) Teknova Q2446 Nuclease-free water AmbionAM9937 Triton X-100 Sigma-Aldrich T8787-100ML RV88 GVK bio DSPC Lipoid556500 Cholesterol Sigma C3045-5G PEG2K Avanti Polar Lipids 880150Ethanol Acros Organic 615090010 5 mL Borosilicate glass vials ThermoScientific ST5-20 PD MiniTrap G-25 Desalting GE Healthcare VWR Cat.Columns #95055-984 Quant-IT RiboGreen RNA Assay Molecular Probes/ R11490kit Life Technologies Black 96-well microplates Greiner 655900

RV88, DSPC, and cholesterol all being prepared in ethanol at aconcentration of 10 mg/ml in borosilica vials. The lipid 14:0-PEG2K PEis prepared at a concentration of 4 mg/ml also in a borosilica glassvial. Dissolution of lipids at stock concentrations is attained bysonication of the lipids in ethanol for 2 min. The solutions are thenheated on an orbital tilting shaker set at 170 rpm at 37° C. for 10 min.Vials are then equilibrated at 26° C. for a minimum of 45 min. Thelipids are then mixed by adding volumes of stock lipid as shown in Table32b. The solution is then adjusted with ethanol such that the finallipid concentration was 7.92 mg/ml.

TABLE 32b Stock Ethanol Composition MW % nmoles mg (mg/ml) ul (ul) RV88794.2 40% 7200 5.72 10 571.8 155.3 DSPC 790.15 10% 1800 1.42 10 142.2Cholesterol 386.67 48% 8640 3.34 10 334.1 PEG2K 2693.3  2% 380 0.97 4242.4

RNA is prepared as a stock solution with 75 mM Citrate buffer at pH 6.0and a concentration of RNA at 1.250 mg/ml. The concentration of the RNAis then adjusted to 0.1037 mg/ml with 75 mM citrate buffer at pH 6.0,equilibrated to 26° C. The solution is then incubated at 26° C. for aminimum of 25 min.

The microfluidic chamber is cleaned with ethanol and neMYSIS syringepumps are prepared by loading a syringe with the RNA solution andanother syringe with the ethanolic lipid. Both syringes are loaded andunder the control of neMESYS software. The solutions are then applied tothe mixing chip at an aqueous to organic phase ratio of 2 and a totalflow rate of 22 ml/min (14.67 ml/min for RNA and 7.33 ml/min for thelipid solution. Both pumps are started synchronously. The mixer solutionthat flowed from the microfluidic chip is collected in 4×1 ml fractionswith the first fraction being discarded as waste. The remaining solutioncontaining the RNA-liposomes is exchanged by using G-25 mini desaltingcolumns to 10 mM Tris-HCl, 1 mM EDTA, at pH 7.5. Following bufferexchange, the materials are characterized for size, and RNA entrapmentthrough DLS analysis and Ribogreen assays, respectively.

Example 30

RNA Containing Transfer Vehicle Using RV94.

In this example, RNA containing liposome are synthesized using the 2-Dvortex microfluidic chip with the cationic lipid RV94 for delivery ofcircRNA.

TABLE 33 Materials and Instrument Vendor Cat # 1M Tris-HCl, pH 8.0,Sterile Teknova T1080 5M Sodium Chloride solution Teknova S0250 QBCitrate buffer, pH 6.0 (100 mM) Teknova Q2446 Nuclease-free water AmbionAM9937 Triton X-100 Sigma-Aldrich T8787-100ML RV94 GVK bio DSPC Lipoid556500 Cholesterol Sigma C3045-5G PEG2K Avanti Polar Lipids 880150Ethanol Acros Organic 615090010 5 mL Borosilicate glass vials ThermoScientific ST5-20 PD MiniTrap G-25 Desalting GE Healthcare VWR Cat.Columns #95055-984 Quant-IT RiboGreen RNA Assay Molecular Probes/ R11490kit Life Technologies Black 96-well microplates Greiner 655900

The lipids were prepared as in Example 29 using the material amountsnamed in Table 34 to a final lipid concentration of 7.92 mg/ml.

TABLE 34 Stock Ethanol Composition MW % nmoles mg (mg/ml) ul (ul) RV94808.22 40% 2880 2.33 10 232.8 155.3 DSPC 790.15 10% 720 0.57 10 56.9Cholesterol 386.67 48% 3456 1.34 10 133.6 PEG2K 2693.3  2% 144 0.39 497.0

The aqueous solution of circRNA is prepared as a stock solution with 75mM Citrate buffer at pH 6.0 the circRNA at 1.250 mg/ml. Theconcentration of the RNA is then adjusted to 0.1037 mg/ml with 75 mMcitrate buffer at pH 6.0, equilibrated to 26° C. The solution is thenincubated at 26° C. for a minimum of 25 min.

The microfluidic chamber is cleaned with ethanol and neMYSIS syringepumps are prepared by loading a syringe with the RNA solution andanother syringe with the ethanolic lipid. Both syringes are loaded andunder the control of neMESYS software. The solutions are then applied tothe mixing chip at an aqueous to organic phase ratio of 2 and a totalflow rate of 22 ml/min (14.67 ml/min for RNA and 7.33 ml/min for thelipid solution. Both pumps are started synchronously. The mixer solutionthat flowed from the microfluidic chip is collected in 4×1 ml fractionswith the first fraction being discarded as waste. The remaining solutioncontaining the circRNA-transfer vehicles is exchanged by using G-25 minidesalting columns to 10 mM Tris-HCl, 1 mM EDTA, at pH 7.5, as describedabove. Following buffer exchange, the materials are characterized forsize, and RNA entrapment through DLS analysis and Ribogreen assays,respectively. The biophysical analysis of the liposomes is shown inTable 35.

TABLE 35 Ratio RNA RNA (aqueous/ encapsulatiom encapsulatiom Sample NPTFR org amount yield size Name Ratio ml/min phase) (μg/ml) % d · nm PDISAM-RV94 8 22 2 31.46 86.9 113.1 0.12

Example 31

General Protocol for in Line Mixing.

Individual and separate stock solutions are prepared—one containinglipid and the other circRNA. Lipid stock containing a desired lipid orlipid mixture, DSPC, cholesterol and PEG lipid is prepared bysolubilized in 90% ethanol. The remaining 10% is low pH citrate buffer.The concentration of the lipid stock is 4 mg/mL. The pH of this citratebuffer can range between pH 3 and pH 5, depending on the type of lipidemployed. The circRNA is also solubilized in citrate buffer at aconcentration of 4 mg/mL. 5 mL of each stock solution is prepared.

Stock solutions are completely clear and lipids are ensured to becompletely solubilized before combining with circRNA. Stock solutionsmay be heated to completely solubilize the lipids. The circRNAs used inthe process may be unmodified or modified oligonucleotides and may beconjugated with lipophilic moieties such as cholesterol.

The individual stocks are combined by pumping each solution to aT-junction. A dual-head Watson-Marlow pump was used to simultaneouslycontrol the start and stop of the two streams. A 1.6 mm polypropylenetubing is further downsized to 0.8 mm tubing in order to increase thelinear flow rate. The polypropylene line (ID=0.8 mm) are attached toeither side of a T-junction. The polypropylene T has a linear edge of1.6 mm for a resultant volume of 4.1 mm³. Each of the large ends (1.6mm) of polypropylene line is placed into test tubes containing eithersolubilized lipid stock or solubilized circRNA. After the T-junction, asingle tubing is placed where the combined stream exited. The tubing isthen extended into a container with 2× volume of PBS, which is rapidlystirred. The flow rate for the pump is at a setting of 300 rpm or 110mL/min. Ethanol is removed and exchanged for PBS by dialysis. The lipidformulations are then concentrated using centrifugation or diafiltrationto an appropriate working concentration.

C57BL/6 mice (Charles River Labs, MA) receive either saline orformulated circRNA via tail vein injection. At various time points afteradministration, serum samples are collected by retroorbital bleed. Serumlevels of Factor VII protein are determined in samples using achromogenic assay (Biophen FVTI, Aniara Corporation, OH). To determineliver RNA levels of Factor VII, animals are sacrificed and livers areharvested and snap frozen in liquid nitrogen. Tissue lysates areprepared from the frozen tissues and liver RNA levels of Factor VII arequantified using a branched DNA assay (QuantiGene Assay, Panomics, CA).

FVII activity is evaluated in FVTI siRNA-treated animals at 48 hoursafter intravenous (bolus) injection in C57BL/6 mice. FVII is measuredusing a commercially available kit for determining protein levels inserum or tissue, following the manufacturer's instructions at amicroplate scale. FVII reduction is determined against untreated controlmice, and the results are expressed as % Residual FVII. Two dose levels(0.05 and 0.005 mg/kg FVII siRNA) are used in the screen of each novelliposome composition.

Example 32

circRNA Formulation Using Preformed Vesicles.

Cationic lipid containing transfer vehicles are made using the preformedvesicle method. Cationic lipid, DSPC, cholesterol and PEG-lipid aresolubilized in ethanol at a molar ratio of 40/10/40/10, respectively.The lipid mixture is added to an aqueous buffer (50 mM citrate, pH 4)with mixing to a final ethanol and lipid concentration of 30% (vol/vol)and 6.1 mg/mL respectively and allowed to equilibrate at roomtemperature for 2 min before extrusion. The hydrated lipids are extrudedthrough two stacked 80 nm pore-sized filters (Nuclepore) at 22° C. usinga Lipex Extruder (Northern Lipids, Vancouver, BC) until a vesiclediameter of 70-90 nm, as determined by Nicomp analysis, is obtained. Forcationic lipid mixtures which do not form small vesicles, hydrating thelipid mixture with a lower pH buffer (50 mM citrate, pH 3) to protonatethe phosphate group on the DSPC headgroup helps form stable 70-90 nmvesicles.

The FVII circRNA (solubilised in a 50 mM citrate, pH 4 aqueous solutioncontaining 30% ethanol) is added to the vesicles, pre-equilibrated to35° C., at a rate of −5 mL/min with mixing. After a final targetcircRNA/lipid ratio of 0.06 (wt wt) is achieved, the mixture isincubated for a further 30 min at 35° C. to allow vesiclere-organization and encapsulation of the FVII RNA. The ethanol is thenremoved and the external buffer replaced with PBS (155 mM NaCl, 3 mMNa2HPO4, ImM KH2PO4, pH 7.5) by either dialysis or tangential flowdiafiltration. The final encapsulated circRNA-to-lipid ratio isdetermined after removal of unencapsulated RNA using size-exclusion spincolumns or ion exchange spin columns.

Example 33

Expression of Trispecific Antigen Binding Proteins from EngineeredCircular RNA

Circular RNAs are designed to include: (1) a 3′ post splicing group Iintron fragment; (2) an Internal Ribosome Entry Site (IRES); (3) atrispecific antigen-binding protein coding region; and (4) a 3′ homologyregion. The trispecific antigen-binding protein regions are constructedto produce an exemplary trispecific antigen-binding protein that willbind to a target antigen, e.g., GPC3.

Generation of a scFv CD3 Binding Domain

The human CD3epsilon chain canonical sequence is Uniprot Accession No.P07766. The human CD3gamma chain canonical sequence is Uniprot AccessionNo. P09693. The human CD3delta chain canonical sequence is UniprotAccession No. P043234. Antibodies against CD3epsilon, CD3gamma orCD3delta are generated via known technologies such as affinitymaturation. Where murine anti-CD3 antibodies are used as a startingmaterial, humanization of murine anti-CD3 antibodies is desired for theclinical setting, where the mouse-specific residues may induce ahuman-anti-mouse antigen (HAMA) response in subjects who receivetreatment of a trispecific antigen-binding protein described herein.Humanization is accomplished by grafting CDR regions from murineanti-CD3 antibody onto appropriate human germline acceptor frameworks,optionally including other modifications to CDR and/or frameworkregions.

Human or humanized anti-CD3 antibodies are therefore used to generatescFv sequences for CD3 binding domains of a trispecific antigen-bindingprotein. DNA sequences coding for human or humanized VL and VH domainsare obtained, and the codons for the constructs are, optionally,optimized for expression in cells from Homo sapiens. The order in whichthe VL and VH domains appear in the scFv is varied (i.e. VL-VH, or VH-VLorientation), and three copies of the “G4S” or “G₄S” subunit (G₄S)₃connect the variable domains to create the scFv domain. Anti-CD3 scFvplasmid constructs can have optional Flag, His or other affinity tags,and are electroporated into HEK293 or other suitable human or mammaliancell lines and purified. Validation assays include binding analysis byFACS, kinetic analysis using Proteon, and staining of CD3-expressingcells.

Generation of a scFv Glypican-3 (GPC3) Binding Domain

Glypican-3 (GPC3) is one of the cell surface proteins present onHepatocellular Carcinoma but not on healthy normal liver tissue. It isfrequently observed to be elevated in hepatocellular carcinoma and isassociated with poor prognosis for HCC patients. It is known to activateWnt signalling. GPC3 antibodies have been generated including MDX-1414,HN3, GC33, and YP7.

A scFv binding to GPC-3 or another target antigen is generated similarlyto the above method for generation of a scFv binding domain to CD3.

Expression of Trispecific Antigen-Binding Proteins In Vitro

A CHO cell expression system (Flp-In®, Life Technologies), a derivativeof CHO-K1 Chinese Hamster ovary cells (ATCC, CCL-61) (Kao and Puck,Proc. Natl. Acad Sci USA 1968; 60(4):1275-81), is used. Adherent cellsare subcultured according to standard cell culture protocols provided byLife Technologies.

For adaption to growth in suspension, cells are detached from tissueculture flasks and placed in serum-free medium. Suspension-adapted cellsare cryopreserved in medium with 10% DMSO.

Recombinant CHO cell lines stably expressing secreted trispecificantigen-binding proteins are generated by transfection ofsuspension-adapted cells. During selection with the antibioticHygromycin B viable cell densities are measured twice a week, and cellsare centrifuged and resuspended in fresh selection medium at a maximaldensity of 0.1×10⁶ viable cells/mL. Cell pools stably expressingtrispecific antigen-binding proteins are recovered after 2-3 weeks ofselection at which point cells are transferred to standard culturemedium in shake flasks. Expression of recombinant secreted proteins isconfirmed by performing protein gel electrophoresis or flow cytometry.Stable cell pools are cryopreserved in DMSO containing medium.

Trispecific antigen-binding proteins are produced in 10-day fed-batchcultures of stably transfected CHO cell lines by secretion into the cellculture supernatant. Cell culture supernatants are harvested after 10days at culture viabilities of typically >75%. Samples are collectedfrom the production cultures every other day and cell density andviability are assessed. On day of harvest, cell culture supernatants arecleared by centrifugation and vacuum filtration before further use.

Protein expression titers and product integrity in cell culturesupernatants are analyzed by SDS-PAGE.

Purification of Trispecific Antigen-Binding Proteins

Trispecific antigen-binding proteins are purified from CHO cell culturesupernatants in a two-step procedure. The constructs are subjected toaffinity chromatography in a first step followed by preparative sizeexclusion chromatography (SEC) on Superdex 200 in a second step. Samplesare buffer-exchanged and concentrated by ultrafiltration to a typicalconcentration of >1 mg/mL Purity and homogeneity (typically >90%) offinal samples are assessed by SDS PAGE under reducing and non-reducingconditions, followed by immunoblotting using an anti-(half-lifeextension domain) or anti idiotype antibody as well as by analyticalSEC, respectively. Purified proteins are stored at aliquots at −80° C.until use.

Example 34

Expression of Engineered Circular RNA with a Half-Life Extension Domainhas Improved Pharmacokinetic Parameters than without a Half-LifeExtension Domain

The trispecific antigen-binding protein encoded on a circRNA molecule ofexample 23 is administered to cynomolgus monkeys as a 0.5 mg/kg bolusinjection intramuscularly. Another cynomolgus monkey group receives acomparable protein encoded on a circRNA molecule in size with bindingdomains to CD3 and GPC-3, but lacking a half-life extension domain. Athird and fourth group receive a protein encoded on a circRNA moleculewith CD3 and half-life extension domain binding domains and a proteinwith GPC-3 and half-life extension domains, respectively. Both proteinsencoded by circRNA are comparable in size to the trispecificantigen-binding protein. Each test group consists of 5 monkeys. Serumsamples are taken at indicated time points, serially diluted, and theconcentration of the proteins is determined using a binding ELISA to CD3and/or GPC-3.

Pharmacokinetic analysis is performed using the test article plasmaconcentrations. Group mean plasma data for each test article conforms toa multi-exponential profile when plotted against the time post-dosing.The data are fit by a standard two-compartment model with bolus inputand first-order rate constants for distribution and elimination phases.The general equation for the best fit of the data for i.v.administration is: c(t)=Ae^(˜at)+Be^(˜pt), where c(t) is the plasmaconcentration at time t, A and B are intercepts on the Y-axis, and a andβ are the apparent first-order rate constants for the distribution andelimination phases, respectively. The a-phase is the initial phase ofthe clearance and reflects distribution of the protein into allextracellular fluid of the animal, whereas the second or β-phase portionof the decay curve represents true plasma clearance. Methods for fittingsuch equations are well known in the art. For example,A=D/V(a−k21)/(a−p), B=D/V(p−k21)/(a−p), and a and β (for α>β) are rootsof the quadratic equation: r²+(k12+k21+k10)r+k21k10=0 using estimatedparameters of V=volume of distribution, k10=elimination rate,k12=transfer rate from compartment 1 to compartment 2 and k21=transferrate from compartment 2 to compartment 1, and D=the administered dose.

Data analysis: Graphs of concentration versus time profiles are madeusing KaleidaGraph (KaleidaGraph™ V. 3.09 Copyright 1986-1997. SynergySoftware. Reading, Pa.). Values reported as less than reportable (LTR)are not included in the PK analysis and are not represented graphically.Pharmacokinetic parameters are determined by compartmental analysisusing WinNonlin software (WinNonlin® Professional V. 3.1 WinNonlin™Copyright 1998-1999. Pharsight Corporation. Mountain View, Calif.).Pharmacokinetic parameters are computed as described in Ritschel W A andKearns G L, 1999, EST: Handbook Of Basic Pharmacokinetics IncludingClinical Applications, 5th edition, American Pharmaceutical Assoc.,Washington, D C.

It is expected that the trispecific antigen-binding protein encoded on acircRNA molecule of Example 23 has improved pharmacokinetic parameterssuch as an increase in elimination half-time as compared to proteinslacking a half-life extension domain.

Example 35

Cytotoxicity of the Trispecific Antigen-Binding Protein

The trispecific antigen-binding protein encoded on a circRNA molecule ofExample 23 is evaluated in vitro on its mediation of T cell dependentcytotoxicity to GPC-3+ target cells.

Fluorescence labeled GPC3 target cells are incubated with isolated PBMCof random donors or T-cells as effector cells in the presence of thetrispecific antigen-binding protein of Example 23. After incubation for4 h at 37° C. in a humidified incubator, the release of the fluorescentdye from the target cells into the supernatant is determined in aspectrofluorimeter. Target cells incubated without the trispecificantigen-binding protein of Example 23 and target cells totally lysed bythe addition of saponin at the end of the incubation serve as negativeand positive controls, respectively.

Based on the measured remaining living target cells, the percentage ofspecific cell lysis is calculated according to the following formula:[1-(number of living targets (sample)/number of living targets(spontaneous))]×100%. Sigmoidal dose response curves and EC50 values arecalculated by non-linear regression/4-parameter logistic fit using theGraphPad Software. The lysis values obtained for a given antibodyconcentration are used to calculate sigmoidal dose-response curves by 4parameter logistic fit analysis using the Prism software.

Example 36 Synthesis of Ionizable Lipids 38.1 Synthesis of((3-(2-methyl-1H-imidazol-1-yl)propyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)(Lipid 27, Table 10a) and((3-(1H-imidazol-1-yl)propyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)) (Lipid 26, Table 10a)

In a 100 mL round bottom flask connected with condenser,3-(1H-imidazol-1-yl)propan-1-amine (100 mg, 0.799 mmol) or3-(2-methyl-1H-imidazol-1-yl)propan-1-amine (0.799 mmol), 6-bromohexyl2-hexyldecanoate (737.2 mg, 1.757 mmol), potassium carbonate (485 mg,3.515 mmol) and potassium iodide (13 mg, 0.08 mmol) were mixed inacetonitrile (30 mL), and the reaction mixture was heated to 80° C. for48 h. The mixture was cooled to room temperature and was filteredthrough a pad of Celite. The filtrate was diluted with ethyl acetate.After washing with water, brine and dried over anhydrous sodium sulfate.The solvent was evaporated and the crude residue was purified by flashchromatography (SiO₂: CH₂Cl₂=100% to 10% of methanol in CH₂Cl₂) andcolorless oil product was obtained (92 mg, 15%). Molecular formula of((3-(1H-imidazol-1-yl)propyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)) is C₅₀H₉₅N₃O₄ and molecular weight (M_(w)) is801.7.

Reaction Scheme for Synthesis of((3-(1H-imidazol-1-yl)propyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)) (Lipid 26 Table 10a)

Characterization of Lipid 26 was performed by LC-MS. FIG. 27A-C showscharacterization of Lipid 26. FIG. 27A shows the proton NMR observed forLipid 26. FIG. 27B is a representative LC/MS trace for Lipid 26 withtotal ion and UV chromatograms shown.

38.2 Synthesis of Lipid 22-S14 38.2.1 Synthesis of2-(tetradecylthio)ethan-1-ol

To a mixture of 2-sulfanylethanol (5.40 g, 69.11 mmol, 4.82 mL, 0.871eq) in acetonitrile (200 mL) was added 1-Bromotetradecane (22 g, 79.34mmol, 23.66 mL, 1 eq) and potassium carbonate (17.55 g, 126.95 mmol, 1.6eq) at 25° C. The reaction mixture was warmed to 40° C. and stirred for12 hr. TLC (ethyl acetate/petroleum ether=25/1, R_(f)=0.3, stained byI₂) showed the starting material was consumed completely and a new mainspot was generated. The reaction mixture was filtered and the filtercake was washed with acetonitrile (50 mL) and then the filtrate wasconcentrated under vacuum to get a residue which was purified by columnon silica gel (ethyl acetate/petroleum ether=1/100 to 1/25) to afford2-(tetradecylthio)ethan-1-ol (14 g, yield 64.28%) as a white solid.

¹H NMR (ET36387-45-P1A, 400 MHz, CHLOROFORM-d) δ 0.87-0.91 (m, 3H) 1.27(s, 20H) 1.35-1.43 (m, 2H) 1.53-1.64 (m, 2H) 2.16 (br s, 1H) 2.49-2.56(m, 2H) 2.74 (t, J=5.93 Hz, 2H) 3.72 (br d, J=4.89 Hz, 2H). FIG. 28shows corresponding Nuclear Magnetic Resonance (NMR) spectrum.

38.2.2 Synthesis of 2-(tetradecylthio)ethyl acrylate

To a solution of 2-(tetradecylthio)ethan-1-ol (14 g, 51.00 mmol, 1 eq)in dichloromethane (240 mL) was added triethylamine (7.74 g, 76.50 mmol,10.65 mL, 1.5 eq) and prop-2-enoyl chloride (5.54 g, 61.20 mmol, 4.99mL, 1.2 eq) dropwise at 0° C. under nitrogen. The reaction mixture waswarmed to 25° C. and stirred for 12 hr. TLC (ethyl acetate/petroleumether=25/1, Rf=0.5, stained by I₂) showed the starting material wasconsumed completely and a new main spot was generated. The reactionsolution was concentrated under vacuum to get crude which was purifiedby column on silica gel (ethyl acetate/petroleum ether=1/100 to 1/25) toafford 2-(tetradecylthio)ethyl acrylate (12 g, yield 71.61%) as acolorless oil.

¹H NMR (ET36387-49-P1A, 400 MHz, CHLOROFORM-d) δ 0.85-0.93 (m, 3H) 1.26(s, 19H) 1.35-1.43 (m, 2H) 1.53-1.65 (m, 2H) 2.53-2.62 (m, 2H) 2.79 (t,J=7.03 Hz, 2H) 4.32 (t, J=7.03 Hz, 2H) 5.86 (dd, J=10.39, 1.47 Hz, 1H)6.09-6.19 (m, 1H) 6.43 (dd, J=17.30, 1.41 Hz, 1H). FIG. 29 showscorresponding Nuclear Magnetic Resonance (NMR) spectrum.

38.2.3 Synthesis of bis(2-(tetradecylthio)ethyl)3,3′-((3-(2-methyl-1H-imidazol-1-yl)propyl)azanediyl)dipropionate (Lipid22-S14)

A flask was charged with 3-(2-methyl-1H-imidazol-1-yl)propan-1-amine(300 mg, 2.16 mmol) and 2-(tetradecylthio)ethyl acrylate (1.70 g, 5.17mmol). The neat reaction mixture was heated to 80° C. and stirred for 48hr. TLC (ethyl acetate, R_(f)=0.3, stained by I₂, one drop ammoniumhydroxide added) showed the starting material was consumed completelyand a new main spot was formed. The reaction mixture was diluted withdichloromethane (4 mL) and purified by column on silica gel (petroleumether/ethyl acetate=3/1 to 0/1, 0.1% ammonium hydroxide added) to getbis(2-(tetradecylthio)ethyl)3,3′-((3-(2-methyl-1H-imidazol-1-yl)propyl)azanediyl)dipropionate (501mg, yield 29.1%) as colorless oil.

¹H NMR (ET36387-51-P1A, 400 MHz, CHLOROFORM-d) δ 0.87 (t, J=6.73 Hz, 6H)1.25 (s, 40H) 1.33-1.40 (m, 4H) 1.52-1.61 (m, 4H) 1.81-1.90 (m, 2H) 2.36(s, 3H) 2.39-2.46 (m, 6H) 2.53 (t, J=7.39 Hz, 4H) 2.70-2.78 (m, 8H) 3.84(t, J=7.17 Hz, 2H) 4.21 (t, J=6.95 Hz, 4H) 6.85 (s, 1H) 6.89 (s, 1H).FIG. 30 shows corresponding Nuclear Magnetic Resonance (NMR) spectrum.

38.3 Synthesis of bis(2-(tetradecylthio)ethyl)3,3′-((3-(1H-imidazol-1-yl)propyl)azanediyl)dipropionate (Lipid 93-S14)

A flask was charged with 3-(1H-imidazol-1-yl)propan-1-amine (300 mg,2.40 mmol, 1 eq) and 2-(tetradecylthio)ethyl acrylate (1.89 g, 5.75mmol, 2.4 eq). The neat reaction mixture was heated to 80° C. andstirred for 48 hr. TLC (ethyl acetate, R_(f)=0.3, stained by I₂, onedrop ammonium hydroxide added) showed the starting material was consumedcompletely and a new main spot was formed. The reaction mixture wasdiluted with dichloromethane (4 mL) and purified by column on silica gel(petroleum ether/ethyl acetate=1/20-0/100, 0.1% ammonium hydroxideadded) to get bis(2-(tetradecylthio)ethyl)3,3′-((3-(1H-imidazol-1-yl)propyl)azanediyl)dipropionate (512 mg, yield27.22%) as colorless oil.

¹H NMR (ET36387-54-P1A, 400 MHz, CHLOROFORM-d) δ 0.89 (t, J=6.84 Hz, 6H)1.26 (s, 40H) 1.34-1.41 (m, 4H) 1.58 (br t, J=7.50 Hz, 4H) 1.92 (t,J=6.62 Hz, 2H) 2.36-2.46 (m, 6H) 2.55 (t, J=7.50 Hz, 4H) 2.75 (q, J=6.84Hz, 8H) 3.97 (t, J=6.95 Hz, 2H) 4.23 (t, J=6.95 Hz, 4H) 6.95 (s, 1H)7.06 (s, 1H) 7.51 (s, 1H). FIG. 31 shows corresponding Nuclear MagneticResonance (NMR) spectrum.

38.4 Synthesis of heptadecan-9-yl8-((3-(2-methyl-M-imidazol-1-yl)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate(Lipid 54, Table 10a) 38.4.1 Synthesis of nonyl 8-bromooctanoate (3)

To a mixture of 8-bromooctanoic acid (2) (18.6 g, 83.18 mmol) andnonan-1-ol (1) (10 g, 69.32 mmol) in CH₂Cl₂ (500 mL) was added DMAP (1.7g, 13.86 mmol), DIPEA (48 mL, 277.3 mmol) and EDC (16 g, 83.18 mmol).The reaction was stirred at room temperature overnight. Afterconcentration of the reaction mixture, the crude residue was dissolvedin ethyl acetate (500 mL), washed with 1N HCl, sat. NaHCO₃, water andBrine. The organic layer was dried over anhydrous Na₂SO₄. The solventwas evaporated and the crude residue was purified by flashchromatography (SiO₂: Hexane=100% to 30% of EtOAc in Hexane) andcolorless oil product 3 was obtained (9 g, 37%).

38.4.2 Synthesis of heptadecan-9-yl 8-bromooctanoate (5)

To a mixture of 8-bromooctanoic acid (2) (10 g, 44.82 mmol) andheptadecan-9-ol (4) (9.6 g, 37.35 mmol) in CH₂C12 (300 mL) was addedDMAP (900 mg, 7.48 mmol), DIPEA (26 mL, 149.7 mmol) and EDC (10.7 g,56.03 mmol). The reaction was stirred at room temperature overnight.After concentration of the reaction mixture, the crude residue wasdissolved in ethyl acetate (300 mL), washed with 1N HCl, sat. NaHCO₃,water and Brine. The organic layer was dried over anhydrous Na₂SO₄. Thesolvent was evaporated and the crude residue was purified by flashchromatography (SiO₂: Hexane=100% to 30% of EtOAc in Hexane) andcolorless oil product 5 was obtained (5 g, 29%).

38.4.3 Synthesis of heptadecan-9-yl84(3-(2-methyl-1H-imidazol-1-yl)propyl)amino)octanoate (7)

In a 100 mL round bottom flask connected with condenser, heptadecan-9-yl8-bromooctanoate (5) (860 mg, 1.868 mmol) and3-(2-methyl-1H-imidazol-1-yl)propan-1-amine (6) (1.3 g, 9.339 mmol) weremixed in ethanol (10 mL). The reaction mixture was heated to refluxovernight. MS (APCI) showed the expected product. The mixture was cooledto room temperature and concentrated. The crude residue was purified byflash chromatography (SiO₂: CH₂Cl₂=100% to 10% of methanol+1% NH₄OH inCH₂Cl₂) and colorless oil product 7 was obtained (665 mg, 69%).

38.4.4 Synthesis of heptadecan-9-yl84(3-(2-methyl-1H-imidazol-1-yl)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate(Lipid 54, Table 10a)

In a 100 mL round bottom flask connected with condenser, heptadecan-9-yl8-((3-(2-methyl-1H-imidazol-1-yl)propyl)amino)octanoate (7) (665 mg,1.279 mmol) and nonyl 8-bromooctanoate (3) (536 mg, 1.535 mmol) weremixed in ethanol (10 mL), then DIPEA (0.55 mL, 3.198 mmol) was added.The reaction mixture was heated to reflux overnight. Both MS (APCI) andTLC (10% MeOH+1% NH₄OH in CH₂Cl₂) showed the product and some unreactedstarting material. The mixture was cooled to room temperature andconcentrated. The crude residue was purified by flash chromatography(SiO₂: CH₂Cl₂=100% to 10% of methanol+1% NH₄OH in CH₂C12) and colorlessoil was obtained (170 mg, 17%).

38.5 Synthesis of heptadecan-9-yl84(3-(1H-imidazol-1-yl)propyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate(Lipid 53, TABLE 10a)

Lipid 53 from Table 10a is synthesized according to the scheme above.Reaction conditions are identical to Lipid 54 with the exception of3-(1H-imidazol-1-yl)propan-1-amine as the imidazole amine.

Example 37

Lipid Nanoparticle Formulation with Circular RNA

Lipid Nanoparticles (LNPs) were formed using a Precision NanosystemsIgnite instrument with a ‘NextGen’ mixing chamber. Ethanol phasecontained ionizable Lipid 26 from Table 10a, DSPC, Cholesterol, andDSPE-PEG 2000 (Avanti Polar Lipids Inc.) at a weight ratio of 16:1:4:1or 62:4:33:1 molar ratio was combined with an aqueous phase containingcircular RNA and 25 mM sodium acetate buffer at pH 5.2. A 3:1 aqueous toethanol mixing ratio was used. The formulated LNP then were dialyzed in1 L of water and exchanged 2 times over 18 hours. Dialyzed LNPs werefiltered using 0.2 μm filter. Prior to in vivo dosing, LNPs were dilutedin PBS. LNP sizes were determined by dynamic light scattering. A cuvettewith 1 mL of 20 μg/mL LNPs in PBS (pH 7.4) was measured for Z-averageusing the Malvern Panalytical Zetasizer Pro. The Z-average andpolydispersity index were recorded.

39.1 Formulation of Lipids 26 and 27 from Table 10a

Lipid Nanoparticles (LNPs) were formed using a Precision NanosystemsIgnite instrument with a ‘NextGen’ mixing chamber. Ethanol phasecontained ionizable Lipid 26 or Lipid 27 from Table 10a, DOPE,Cholesterol, and DSPE-PEG 2000 (Avanti Polar Lipids Inc.) at a weightratio of 16:1:4:1 or 62:4:33:1 molar ratio was combined with an aqueousphase containing circular RNA and 25 mM sodium acetate buffer at pH 5.2.A 3:1 aqueous to ethanol mixing ratio was used. The formulated LNPs werethen dialyzed in 1 L of water and exchanged 2 times over 18 hours.Dialyzed LNPs were filtered using 0.2 μm filter. Prior to in vivodosing, LNPs were diluted in PBS. LNP sizes were determined by dynamiclight scattering. A cuvette with 1 mL of 20 μg/mL LNPs in PBS (pH 7.4)was measured for Z-average using the Malvern Panalytical Zetasizer Pro.The Z-average and polydispersity index were recorded.

39.2 Formulation of Lipids 53 and 54 from Table 10a

Lipid Nanoparticles (LNPs) were formed using a Precision NanosystemsIgnite instrument with a ‘NextGen’ mixing chamber. Ethanol phasecontained ionizable Lipid 53 or 54 of Table 10a, DOPE, Cholesterol, andDSPE-PEG 2000 (Avanti Polar Lipids Inc.) at a molar ratio of50:10:38.5:1.5 was combined with an aqueous phase containing circularRNA and 25 mM sodium acetate buffer at pH 5.2. A 3:1 aqueous to ethanolmixing ratio was used. The formulated LNPs were then dialyzed in 1 L of1×PBS and exchanged 2 times over 18 hours. Dialyzed LNPs were filteredusing 0.2 μm filter. Prior to in vivo dosing, LNPs were diluted in PBS.LNP sizes were determined by dynamic light scattering. A cuvette with 1mL of 20 μg/mL LNPs in PBS (pH 7.4) was measured for Z-average using theMalvern Panalytical Zetasizer Pro. The Z-average and polydispersityindex were recorded.

LNP zeta potential was measured using the Malvern Panalytical ZetasizerPro. A mixture containing 200 μL of the particle solution in water and800 μL of distilled RNAse-free water with a final particle concentrationof 400 μg/mL was loaded into a zetasizer capillary cell for analysis.

RNA encapsulation was determined using a Ribogreen assay. Nanoparticlesolutions were diluted in tris-ethylenediaminetetraacetic acid (TE)buffer at a theoretical oRNA concentration of 2 μg/mL. Standard oRNAsolutions diluted in TE buffer were made ranging from 2 μg/mL to 0.125μg/mL. The particles and standards were added to all wells and a secondincubation was performed (37° C. at 350 rpm for 3 minutes). Fluorescencewas measured using a SPECTRAmax® GEMINI XS microplatespectrofluorometer. The concentration of circular RNA in each particlesolution was calculated using the standard curve. The encapsulationefficiency was calculated from the ratio of oRNA detected between lysedand unlysed particles.

TABLE 36a Characterization of LNPs Encapsulation Zeta Data IonizableLipid Size (nm) PDI Efficiency (%) Potential (mV) 22-S14 88 0.09 963.968 93-S14 119 0.02 96 −6.071 Lipid 26, Table 10a 86 0.08 92 −15.24

TABLE 36b Characterization of LNPs RNA Ionizable Lipid Z-Average(nm) PDIEntrapment(%) 22-S14 64 0.05 97 93-S14 74 0.04 95 Lipid 26, Table 10a 840.04 96

Example 38

In Vivo Analysis

Female CD-1 or female c57BL/6J_mice ranging from 22-25 g were dosed at0.5 mg/kg RNA intravenously. Six hours after injection, mice wereinjected intraperitoneally with 200 μL of D-luciferin at 15 mg/mLconcentration. 5 minutes after injection, mice were anesthetized usingisoflurane, and placed inside the IVIS Spectrum In Vivo Imaging System(Perkin Elmer) with dorsal side up. Whole body total IVIS flux of Lipids22-S14, 93-S14, Lipid 26 (Table 10a) is presented in FIG. 32A. Post 10minutes injection, mice were scanned for luminescence. Mice wereeuthanized and organs were extracted within 25 minutes of luciferininjection to scan for luminescence in liver, spleen, kidneys, lungs, andheart. Images (FIGS. 33A-B, 34A-B, 35A-B) were analyzed using LivingImages (Perkin ELMER) software. Regions of interest were drawn to obtainflux and average radiance and analyzed for biodistribution of proteinexpression (FIG. 32A-B).

FIG. 32A illustrates the increased whole-body total flux observed fromluciferase oRNA with Lipid 26 (Table 10a) LNPs compared to LNPs madewith lipids 22-S14 and 93-S14. FIG. 32B shows the ex vivo IVIS analysisof tissues further highlighting the increased overall expression withLipid 26 (Table 10a) while maintaining the desired spleen to liverratios observed with lipids 22-S14 and 93-S14 despite the significantstructural changes designed to improve expression. These data highlightthe improvements afforded by Lipid 26 (Table 10a) compared to previouslyreported lipids.

Similar analysis as described above was also performed with oRNAencapsulated in LNPs formed with Lipid 15 from Table 10b or Lipid 53 or54 from Table 10a. FIGS. 36A-C show the ex vivo IVIS analysis oftissues, respectively highlighting the overall expression with Lipid 15,53, and 54 while maintaining the desired spleen to liver ratios despitethe significant structural changes designed to improve expression. FIG.36D shows the results for PBS control. These data demonstrates theimprovements afforded by Lipids 15, 53, and 54 from Table 10a comparedto previously reported lipids such as 93-S14 and 22-S14.

Example 39

Delivery of Luciferase

Human peripheral blood mononuclear cells (PBMCs) (Stemcell Technologies)were transfected with lipid nanoparticles (LNP) encapsulating fireflyluciferase (f.luc) circular RNA and examined for luciferase expression.PBMCs from two different donors were incubated in vitro with fivedifferent LNP compositions, containing circular RNA encoding for fireflyluciferase (200 ng), at 37° C. in RPMI, 2% human serum, IL-2 (10 ng/mL),and 50 uM BME. PBMCs incubated without LNP were used as a negativecontrol. After 24 hours, the cells were lysed and analyzed for fireflyluciferase expression based on bioluminescence (Promega BrightGlo).

Representative data are presented in FIGS. 37A and 37B, showing thatthat the tested LNPs are capable of delivering circular RNA into primaryhuman immune cells resulting in protein expression.

Example 40

In Vitro Delivery of Green Fluorescent Protein (GFP) or Chimeric AntigenReceptor (CAR)

Human PBMCs (Stemcell Technologies) were transfected with LNPencapsulating GFP and examined by flow cytometry. PBMCs from fivedifferent donors (PBMC A-E) were incubated in vitro with one LNPcomposition, containing circular RNA encoding either GFP or CD19-CAR(200 ng), at 37° C. in RPMI, 2% human serum, IL-2 (10 ng/mL), and 50 uMBME. PBMCs incubated without LNP were used as a negative control. After24, 48, or 72 hours post-LNP incubation, cells were analyzed for CD3,CD19, CD56, CD14, CD11b, CD45, fixable live dead, and payload (GFP orCD19-CAR).

Representative data are presented in FIGS. 38A and 38B, showing that thetested LNP is capable of delivering circular RNA into primary humanimmune cells resulting in protein expression.

Example 41

Multiple IRES Variants can Mediate Expression of Murine CD19 CAR InVitro

Multiple circular RNA constructs, encoding anti-murine CD19 CAR,contains unique IRES sequences and were lipotransfected into 1C1C7 celllines. Prior to lipotransfection, 1C1C7 cells are expanded for severaldays in complete RPMI Once the cells expanded to appropriate numbers,1C1C7 cells were lipotransfected (Invitrogen RNAiMAX) with fourdifferent circular RNA constructs. After 24 hours, 1C1C7 cells wereincubated with His-tagged recombinant murine CD19 (Sino Biological)protein, then stained with a secondary anti-His antibody. Afterwards,the cells were analyzed via flow cytometry.

Representative data are presented in FIG. 39 , showing that IRES sourcedfrom the indicated virus (apodemus agrarius picornavirus, caprinekobuvirus, parabovirus, and salivirus) are capable of driving expressionof an anti-mouse CD19 CAR in murine T cells.

Example 42

Murine CD19 CAR Mediates Tumor Cell Killing In Vitro

Circular RNA encoding anti-mouse CD19 CAR were electroporated intomurine T cells to evaluate CAR-mediated cytotoxicity. Forelectroporation, T cells were electroporated with circular RNA encodinganti-mouse CD19 CAR using ThermoFisher's Neon Transfection System thenrested overnight. For the cytotoxicity assay, electroporated T cellswere co-cultured with Fluc+ target and non-target cells at 1:1 ratio incomplete RPMI containing 10% FBS, IL-2 (10 ng/mL), and 50 uM BME andincubated overnight at 37° C. Cytotoxicity was measured using aluciferase assay system 24 hours post-co-culture (Promega BrightgloLuciferase System) to detect lysis of Fluc+ target and non-target cells.Values shown are calculated relative to the untransfected mock signal.

Representative data are presented in FIG. 40 , showing that ananti-mouse CD19 CAR expressed from circular RNA is functional in murineT cells in vitro.

Example 43

Functional Depletion of B Cells with a Lipid Encapsulated Circular RNAEncoding Murine CD19 CAR

C57BL/6J mice were injected with LNP formed with Lipid 15 in Table 10b,encapsulating circular RNA encoding anti-murine CD19 CAR. As a control,Lipid 15 in Table 10b encapsulating circular RNA encoding fireflyluciferase (f.Luc) were injected in different group of mice. FemaleC57BL.6J, ranging from 20-25 g, were injected intravenously with 5 dosesof 0.5 mg/kg of LNP, every other day. Between injections, blood drawswere analyzed via flow cytometry for fixable live/dead, CD45, TCRvb,B220, CD11b, and anti-murine CAR. Two days after the last injection,spleens were harvested and processed for flow cytometry analysis.Splenocytes were stained with fixable live/dead, CD45, TCRvb, B220,CD11b, NK1.1, F4/80, CD11c, and anti-murine CAR. Data from mice injectedwith anti-murine CD19 CAR LNP were normalized to mice that receivedf.Luc LNP.

Representative data are presented in FIGS. 41A, 41B, and 41C, showingthat an anti-mouse CD19 CAR expressed from circular oRNA delivered invivo with LNPs is functional in murine T cells in vivo.

Example 44

CD19 CAR Expressed from Circular RNA has Higher Yield and GreaterCytotoxic Effect Compared to that Expressed from mRNA

Circular RNA encoding anti-CD19 chimeric antigen receptor, whichincludes, from N-terminus to C-terminus, a FMC63-derived scFv, a CD8transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζintracellular domain, were electroporated into human peripheral T cellsto evaluate surface expression and CAR-mediated cytotoxicity. Forcomparison, circular RNA-electroporated T cells were compared tomRNA-electroporated T cells in this experiment. For electroporation,CD3+ T cells were isolated from human PBMCs using commercially availableT cell isolation kits (Miltenyi Biotec) from donor human PBMCs. Afterisolation, T cells were stimulated with anti-CD3/anti-CD28 (StemcellTechnologies) and expanded over 5 days at 37° C. in complete RPMIcontaining 10% FBS, IL-2 (10 ng/mL), and 50 uM BME. Five days poststimulation, T cells were electroporated with circular RNA encodinganti-human CD19 CAR using ThermoFisher's Neon Transfection System andthen rested overnight. For the cytotoxicity assay, electroporated Tcells were co-cultured with Fluc+ target and non-target cells at 1:1ratio in complete RPMI containing 10% FBS, IL-2 (10 ng/mL), and 50 uMBME and incubated overnight at 37° C. Cytotoxicity was measured using aluciferase assay system 24 hours post-co-culture (Promega BrightgloLuciferase System) to detect lysis of Fluc+ target and non-target cells.Furthermore, an aliquot of electroporated T cells were taken and stainedfor live dead fixable staining, CD3, CD45, and chimeric antigenreceptors (FMC63) at the day of analysis.

Representative data are presented in FIGS. 42 and 43 . FIGS. 42A and 42Bshow that an anti-human CD19 CAR expressed from circular RNA isexpressed at higher levels and longer than an anti-human CD19 CARexpressed from linear mRNA. FIGS. 43A and 43B show that an anti-humanCD19 CAR expressed from circular RNA is exerts a greater cytotoxiceffect relative to anti-human CD19 CAR expressed from linear mRNA.

Example 45

Functional Expression of Two CARs from a Single Circular RNA

Circular RNA encoding chimeric antigen receptors were electroporatedinto human peripheral T cells to evaluate surface expression andCAR-mediated cytotoxicity. The purpose of this study is to evaluate ifcircular RNA encoding for two CARs can be stochastically expressed witha 2A (P2A) or an IRES sequence. For electroporation, CD3+ T cells werecommercially purchased (Cellero) and stimulated with anti-CD3/anti-CD28(Stemcell Technologies) and expanded over 5 days at 37° C. in completeRPMI containing 10% FBS, IL-2 (10 ng/mL), and 50 uM BME. Four days poststimulation, T cells were electroporated with circular RNA encodinganti-human CD19 CAR, anti-human CD19 CAR-2A-anti-human BCMA CAR, andanti-human CD19 CAR-IRES-anti-human BCMA CAR using ThermoFisher's NeonTransfection System then rested overnight. For the cytotoxicity assay,electroporated T cells were co-cultured with Fluc+ K562 cells expressinghuman CD19 or BCMA antigens at 1:1 ratio in complete RPMI containing 10%FBS, IL-2 (10 ng/mL), and 50 uM BME and incubated overnight at 37° C.Cytotoxicity was measured using a luciferase assay system 24 hourspost-co-culture (Promega BrightGlo Luciferase System) to detect lysis ofFluc+ target cells.

Representative data are presented in FIG. 44 , showing that two CARs canbe functionally expressed from the same circular RNA construct and exertcytotoxic effector function.

Example 46

In Vivo Circular RNA Transfection Using Cre Reporter Mice

Circular RNAs encoding Cre recombinase (Cre) are encapsulated into lipidnanoparticles as previously described. Female, 6-8 week oldB6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J (Ai9) mice were dosed withlipid nanoparticles at 0.5 mg/kg RNA intravenously. Fluorescent tdTomatoprotein was transcribed and translated in Ai9 mice upon Crerecombination, meaning circular RNAs have been delivered to andtranslated in tdTomato+ cells. After 48 hr, mice were euthanized and thespleens were harvested, processed into a single cell suspension, andstained with various fluorophore-conjugated antibodies forimmunophenotyping via flow cytometry.

FIG. 45A shows representative FACS plots with frequencies of tdTomatoexpression in various spleen immune cell (CD45+, live) subsets,including total myeloid (CD11b+), B cells (CD19+), and T cells (TCR-B+)following treatment with LNPs formed with Lipid 27 or 26 from Table 10aor Lipid 15 from Table 10b. Ai9 mice injected with PBS representedbackground tdTomato fluorescence. FIG. 45B quantifies the proportion ofmyeloid cells, B cells, and T cells expressing tdTomato (mean+std. dev.,n=3), which is equivalent to the proportion of each cell populationwhich has been successfully transfected with Cre circular RNA. LNPs madewith Lipids 27 and 26 from Table 10a exhibit significantly highermyeloid and T cell transfection compared with Lipid 93-S14, highlightingthe improvements conferred by lipid structural modifications.

FIG. 45C illustrates the proportion of additional splenic immune cellpopulations expressing tdTomato with Lipids 27 and 26 from Table 10a(mean+std. dev., n=3), which also include NK cells (NKp46+, TCR-B−),classical monocytes (CD11b+, Ly-6G-, Ly-6C_hi), nonclassical monocytes(CD11b+, Ly-6G-, Ly-6C_lo), neutrophils (CD11b+, Ly-6G+), and dendriticcells (CD11c+, MHC-II+). These experiments demonstrate that LNPs madewith Lipids 27 and 26 from Table 10a and Lipid 15 from Table 10b areeffective at delivering circular RNAs to many splenic immune cellsubsets in mice and lead to successful protein expression from thecircular RNA in those cells.

Example 47 Example 47A: Built-In polyA Sequences andAffinity-Purification to Produce Immune-Silent Circular RNA

PolyA sequences (20-30 nt) were inserted into the 5′ and 3′ ends of theRNA construct (precursor RNA with built-in polyA sequences in theintrons). Precursor RNA and introns can alternatively be polyadenylatedpost-transcriptionally using, e.g., E coli. polyA polymerase or yeastpolyA polymerase, which requires the use of an additional enzyme.

Circular RNA in this example was circularized by in vitro transcription(IVT) and affinity-purified by washing over a commercially availableoligo-dT resin to selectively remove polyA-tagged sequences (includingfree introns and precursor RNA) from the splicing reaction. The IVT wasperformed with a commercial IVT kit (New England Biolabs) or acustomerized IVT mix (Orna Therapeutics), containing guanosinemonophosphate (GMP) and guanosine triphosphate (GTP) at different ratios(GMP:GTP=8, 12.5, or 13.75). In some embodiments, GMP at a high GMP:GTPratio may be preferentially included as the first nucleotide, yielding amajority of monophosphate-capped precursor RNAs. As a comparison, thecircular RNA product was alternatively purified by the treatment withXrn1, Rnase R, and Dnase I (enzyme purification).

Immunogenicity of the circular RNAs prepared using the affinitypurification or enzyme purification process were then assessed. Briefly,the prepared circular RNAs were transfected into A549 cells. After 24hours, the cells were lysed and interferon beta-1 induction relative tomock samples was measured by qPCR. 3p-hpRNA, a triphosphorylated RNA,was used as a positive control.

FIGS. 46B and 46C show that the negative selection affinity purificationremoves non-circular products from splicing reactions when polyAsequences are included on elements that are removed during splicing andpresent in unspliced precursor molecules. FIG. 46D shows circular RNAsprepared with tested IVT conditions and purification methods are allimmunoquiescent. These results suggest the negative selection affinitypurification is equivalent or superior to enzyme purification forcircular RNA purification and that customized circular RNA synthesisconditions (IVT conditions) may reduce the reliance on GMP excess toachieve maximal immunoquiescence.

Example 47B: Dedicated Binding Site and Affinity Purification forCircular RNA Production

Instead of polyA tags, one can include specifically design sequences(DBS, dedicated binding site).

Instead of a polyA tag, a dedicated binding site (DBS), such as aspecifically designed complementary oligonucleotide that can bind to aresin, may be used to selectively deplete precursor RNA and freeintrons. In this example, DBS sequences (30 nt) were inserted into the5′ and 3′ ends of the precursor RNA. RNA was transcribed and thetranscribed product was washed over a custom complementaryoligonucleotide linked to a resin.

FIGS. 47B and 47C demonstrates that including the designed DBS sequencein elements that are removed during splicing enables the removal ofunspliced precursor RNA and free intron components in a splicingreaction, via negative affinity purification.

Example 47C: Production of a Circular RNA Encoding Dystrophin

A 12 kb 12,000 nt circular RNA encoding dystrophin was produced by invitro transcription of RNA precursors followed by enzyme purificationusing a mixture of Xrn1, DNase 1, and RNase R to degrade remaininglinear components. FIG. 48 shows that the circular RNA encodingdystrophin was successfully produced.

Example 48

5′ Spacer Between 3′ Intron Fragment and the IRES Improves Circular RNAExpression

Expression level of purified circRNAs with different 5′ spacers betweenthe 3′ intron fragment and the IRES in Jurkat cells were compared.Briefly, luminescence from secreted Gaussia luciferase in supernatantwas measured 24 hours after electroporation of 60,000 cells with 250 ngof each RNA.

Additionally, stability of purified circRNAs with different 5′ spacersbetween the 3′ intron fragment and the IRES in Jurkat cells werecompared. Briefly, luminescence from secreted Gaussia luciferase insupernatant was measured over 2 days after electroporation of 60,000cells with 250 ng of each RNA and normalized to day 1 expression.

The results are shown in FIGS. 49A and 49B, indicating that adding aspacer can enhance IRES function and the importance of sequence identityand length of the added spacer. A potential explanation is that thespacer is added right before the IRES and likely functions by allowingthe IRES to fold in isolation from other structured elements such as theintron fragments.

Example 49

This example describes deletion scanning from 5′ or 3′ end of thecaprine kobuvirus IRES. IRES borders are generally poorly characterizedand require empirical analysis, and this example can be used forlocating the core functional sequences required for driving translation.Briefly, circular RNA constructs were generated with truncated IRESelements operably linked to a Gaussia luciferase coding sequence. Thetruncated IRES elements had nucleotide sequences of the indicatedlengths removed from the 5′ or 3′ end. Luminescence from secretedGaussia luciferase in supernatant was measured 24 and 48 hours afterelectroporation of primary human T cells with RNA. Stability ofexpression was calculated as the ratio of the expression level at the48-hour time point relative to that at the 24-hour time point.

As shown in FIG. 50 , deletion of more than 40 nucleotides from the 5′end of the IRES reduced expression and disrupted IRES function.Stability of expression was relatively unaffected by the truncation ofthe IRES element but expression level was substantially reduced bydeletion of 141 nucleotides from the 3′ end of the IRES, whereasdeletion of 57 or 122 nucleotides from the 3′ end had a positive impacton the expression level.

It was also observed that deletion of the 6-nucleotide pre-startsequence reduced the expression level of the luciferase reporter.Replacement of the 6-nucleotide sequence with a classical kozak sequence(GCCACC) did not have a significant impact but at least maintainedexpression.

Example 50

This example describes modifications (e.g., truncations) of selectedIRES sequences, including Caprine Kobuvirus (CKV) IRES, ParabovirusIRES, Apodemus Picornavirus (AP) IRES, Kobuvirus SZAL6 IRES, CrohivirusB (CrVB) IRES, CVB3 IRES, and SAFV IRES. The sequences of the IRESelements are provided in SEQ ID NOs: 348-389. Briefly, circular RNAconstructs were generated with truncated IRES elements operably linkedto a Gaussia luciferase coding sequence. HepG2 cells were transfectedwith the circular RNAs. Luminescence in the supernatant was assessed 24and 48 hours after transfection. Stability of expression was calculatedas the ratio of the expression level at the 48-hour time point relativeto that at the 24-hour time point.

As shown in FIG. 51 , truncations had variable effects depending on theidentity of the IRES, which may depend on the initiation mechanism andprotein factors used for translation, which often differs between IRESs. 5′ and 3′ deletions can be effectively combined, for example, in thecontext of CKV IRES. Addition of a canonical Kozak sequence in somecases significantly improved expression (as in SAFV, Full vs Full+K) ordiminished expression (as in CKV, 5d40/3d122 vs 5d40/3d122+K).

Example 51

This example describes modifications of CK-739, AP-748, and PV-743 IRESsequences, including mutations alterative translation initiation sites.Briefly, circular RNA constructs were generated with modified IRESelements operably linked to Gaussia luciferase coding sequence.Luminescence from secreted Gaussia luciferase in supernatant wasmeasured 24 and 48 hours after transfection of 1C1C7 cells with RNA.

CUG was the most commonly found alternative start site but many otherswere also characterized. These triplets can be present in the IRESscanning tract prior to the start codon and can affect translation ofcorrect polypeptides. Four alternative start site mutations werecreated, with the IRES sequences provided in SEQ ID NOs: 378-380. Asshown in FIG. 52 , mutations of alternative translation initiation sitesin the CK-739 IRES affected translation of correct polypeptides,positively in some instances and negatively in other instances. Mutationof all the alternative translation initiation sites reduced the level oftranslation.

Alternative Kozak sequences, 6 nucleotides before start codon, can alsoaffect expression levels. The 6-nucleotide sequence upstream of thestart codon were gTcacG, aaagtc, gTcacG, gtcatg, gcaaac, and acaacc,respectively, in CK-739 IRES and Sample Nos. 1-5 in the “6 nt Pre-Start”group. As shown in FIG. 52 , substitution of certain 6-nucleotidesequences prior to the start codon affected translation.

It was also observed that 5′ and 3′ terminal deletions in AP-748 andPV-743 IRES sequences reduced expression. However, in the CK-739 IRES,which had a long scanning tract, translation was relatively unaffectedby deletions in the scanning tract.

Example 52

This example describes modifications of selected IRES sequences byinserting 5′ and/or 3′ untranslated regions (UTRs) and creating IREShybrids. Briefly, circular RNA constructs were generated with modifiedIRES elements operably linked to a Gaussia luciferase coding sequence.Luminescence from secreted Gaussia luciferase in supernatant wasmeasured 24 and 48 hours after transfection of HepG2 cells with RNA.

IRES sequences with UTRs inserted are provided in SEQ ID NOs: 390-401.As shown in FIG. 53 , insertion of 5′ UTR right after the 3′ end of theIRES and before the start codon slightly increased the translation fromCaprine Kobuvirus (CK) IRES but in some instances abrogated translationfrom Salivirus SZ1 IRES. Insertion of 3′ UTR right after the stopcassette had no impact on both IRES sequences.

Hybrid CK IRES sequences are provided in SEQ ID NOs: 390-401. CK IRESwas used as a base, and specific regions of the CK IRES were replacedwith similar-looking structures from other IRES sequences, for example,SZ1 and AV (Aichivirus). As shown in FIG. 53 , certain hybrid syntheticIRES sequences were functional, indicating that hybrid IRES can beconstructed using parts from distinct IRES sequences that show similarpredicted structures while deleting these structures completelyabrogates IRES function.

Example 53

This example describes modifications of circular RNAs by introducingstop codon or cassette variants. Briefly, circular RNA constructs weregenerated with IRES elements operably linked to a Gaussia luciferasecoding sequence followed by variable stop codon cassettes, whichincluded a stop codon in each frame and two stop codons in the readingframe of the Gaussia luciferase coding sequence. 1C1C7 cells weretransfected with the circular RNAs. Luminescence in supernatant wasassessed 24 and 48 hours after transfection.

The sequences of the stop codon cassettes are set forth in SEQ ID NOs:406-412. As shown in FIG. 54 , certain stop codon cassettes improvedexpression levels, although they had little impact on expressionstability. In particular, a stop cassette with two frame 1 (the readingframe of the Gaussia luciferase coding sequence) stop codons, the firstbeing TAA, followed by a frame 2 stop codon and a frame 3 stop codon, iseffective for promoting functional translation.

Example 54

This example describes modifications of circular RNAs by inserting 5′UTR variants. Briefly, circular RNA constructs were generated with IRESelements with 5′ UTR variants inserted between the 3′ end of the IRESand the start codon, the IRES being operably linked to a Gaussialuciferase coding sequence. 1C1C7 cells were transfected with thecircular RNAs. Luminescence in supernatant was assessed 24 and 48 hoursafter transfection.

The sequences of the 5′ UTR variants are set forth in SEQ ID NOs:402-405. As shown in FIG. 55 , a CK IRES with a canonical Kozak sequence(UTR4) was more effective when a 36-nucleotide unstructured/low GCspacer sequence was added (UTR2), suggesting that the GC-rich Kozaksequences may interfere with core IRES folding. Using ahigher-GC/structured spacer with a kozak sequence did not show the samebenefit (UTR3), possibly due to interference with IRES folding by thespacer itself. Mutating the kozak sequence to gTcacG (UTR1) enhancedtranslation to the same level as the Kozak+spacer alternative withoutthe need for a spacer.

Example 55

This example describes the impact of miRNA target sites in circular RNAson expression levels. Briefly, circular RNA constructs were generatedwith IRES elements operably linked to a human erythropoietin (hEPO)coding sequence, where 2 tandem miR-122 target sites were inserted intothe construct. miR-122-expressing Huh7 cells were transfected with thecircular RNAs. hEPO expression in supernatant was assessed 24 and 48hours after transfection by sandwich ELISA.

As shown in FIG. 56 , the hEPO expression level was obrogated where themiR-122 target sites were inserted into the circular RNA. This resultdemonstrates that expression from circular RNA can be regulated bymiRNA. As such, cell type- or tissue-specific expression can be achievedby incorporating target sites of the miRNAs expressed in the cell typesin which expression of the recombinant protein is undesirable.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated as being incorporated byreference herein.

What is claimed is:
 1. A pharmaceutical composition comprising: a. acircular RNA polynucleotide comprising (i) a 3′ post-splicing intronsequence of a 3′ group I intron fragment, (ii) an internal ribosomeentry site (IRES), (iii) an expression sequence, and (iv) a 5′post-splicing intron sequence of a 5′ group I intron fragment, and b. anin vivo transfer vehicle comprising an ionizable lipid represented byFormula (5):

wherein: R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀alkenyl, —R*YR″, —YR″, and —R″M′R′; R₂ and R₃ are independently selectedfrom the group consisting of H, C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″,—YR″, and —R*OR″, or R₂ and R₃, together with the atom to which they areattached, form a heterocycle or carbocycle; R₄ is selected from thegroup consisting of a C₃₋₆ carbocycle, —(CH₂)_(n)Q, —(CH₂)_(n)CHQR,—CHQR, —CQ(R)₂, and unsubstituted C₁₋₆ alkyl, where Q is selected from acarbocycle, heterocycle, —OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃,—CX₂H, —CXH₂, —CN, —N(R)₂, —C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R,—N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂, —N(R)R₈, —O(CH₂)_(n)OR,—N(R)C(═NR₉)N(R)₂, —N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR,—N(OR)C(O)R, —N(OR)S(O)₂R, —N(OR)C(O)OR, —N(OR)C(O)N(R)₂,—N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂, —N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)N(R)₂,—C(═NR₉)R, —C(O)N(R)OR, and —C(R)N(R)₂C(O)OR, and each n isindependently selected from 1, 2, 3, 4, and 5; each R₅ is independentlyselected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H; M and M′ are independently selected from—C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—,—SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O)_(2—), —S—S—, an aryl group, and aheteroaryl group; R₇ is selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H; R₈ is selected from the group consisting ofC₃₋₆ carbocycle and heterocycle; R₉ is selected from the groupconsisting of H, —CN, —NO₂, C₁₋₆ alkyl, —OR, —S(O)₂R, —S(O)₂N(R)₂, C₂₋₆alkenyl, C₃₋₆ carbocycle and heterocycle; each R is independentlyselected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H; each R″ is independentlyselected from the group consisting of C₃₋₁₄ alkyl and C₃₋₁₄ alkenyl;each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl; each Y is independently a C₃₋₆ carbocycle; eachX is independently selected from the group consisting of F, Cl, Br, andI; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; and whereinwhen R₄ is —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, or —CQ(R)₂, then (i) Q isnot —N(R)₂ when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or7-membered heterocycloalkyl when n is 1 or
 2. 2. The pharmaceuticalcomposition of claim 1, wherein the expression sequence encodes achimeric antigen receptor (CAR).
 3. The pharmaceutical composition ofclaim 1, wherein the ionizable lipid is represented by Formula (5A):

wherein: l is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6,7, 8, and 9; M₁ is a bond or M′; R₄ is unsubstituted C₁₋₃ alkyl, or—(CH₂)_(n)Q, in which Q is —OH, —NHC(S)N(R)₂, —NHC(O)N(R)₂, —N(R)C(O)R,—N(R)S(O)₂R, —N(R)R₈, —NHC(═NR₉)N(R)₂, —NHC(═CHR₉)N(R)₂, —OC(O)N(R)₂,—N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R, —N(OR)C(O)OR, —N(OR)C(O)N(R)₂,—N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂, —N(OR)C(═CHR₉)N(R)₂, heteroaryl orheterocycloalkyl; M and M′ are independently selected from —C(O)O—,—OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, —S—S—, an aryl group, and aheteroaryl group; and R₂ and R₃ are independently selected from thegroup consisting of H, C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl.
 4. Thepharmaceutical composition of claim 3, wherein the ionizable lipid isrepresented by Formula (5C):


5. The pharmaceutical composition of claim 3, wherein the ionizablelipid is:


6. The pharmaceutical composition of claim 2, comprising a targetingmoiety operably connected to the in vivo transfer vehicle.
 7. Thepharmaceutical composition of claim 1, wherein the composition has aduration of therapeutic effect in vivo in human greater than that of acomposition comprising a reference linear RNA polynucleotide having thesame expression sequence as the circular RNA polynucleotide.
 8. Apharmaceutical composition comprising: a. a circular RNA polynucleotidecomprising (i) a 3′ post-splicing intron sequence of a 3′ group I intronfragment, (ii) an internal ribosome entry site (IRES), (iii) anexpression sequence, and (iv) a 5′ post-splicing intron sequence of a 5′group I intron fragment, wherein the 3′ post-splicing intron sequence ofa 3′ group I intron fragment and 5′ post-splicing intron sequence of a5′ group I intron fragment each individually comprise a nucleotide of asplice site dinucleotide of group I intron fragment and optionally, anexon fragment, and b. an in vivo transfer vehicle comprising anionizable lipid represented by Formula (5A):

wherein: l is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6,7, 8, and 9; M₁ is a bond or M′; R₄ is unsubstituted C₁₋₃ alkyl, or—(CH₂)_(n)Q, in which Q is —OH, —NHC(S)N(R)₂, —NHC(O)N(R)₂, —N(R)C(O)R,—N(R)S(O)₂R, —N(R)R₈, —NHC(═NR₉)N(R)₂, —NHC(═CHR₉)N(R)₂, —OC(O)N(R)₂,—N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R, —N(OR)C(O)OR, —N(OR)C(O)N(R)₂,—N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂, —N(OR)C(═CHR₉)N(R)₂, heteroaryl orheterocycloalkyl; M and M′ are independently selected from —C(O)O—,—OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, —S—S—, an aryl group, and aheteroaryl group; and R₂ and R₃ are independently selected from thegroup consisting of H, C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl.
 9. Thepharmaceutical composition of claim 8, wherein the expression sequenceencodes for a chimeric antigen receptor (CAR).
 10. The pharmaceuticalcomposition of claim 8, wherein the ionizable lipid is:


11. The pharmaceutical composition of claim 8, wherein the RNApolynucleotide comprises a first spacer before the post-splicing intronfragment of the 3′ group I intron fragment, and a second spacer afterthe post-splicing intron fragment of the 5′ group I intron fragment. 12.The pharmaceutical composition of claim 8, wherein the RNApolynucleotide comprises a poly A sequence.
 13. The pharmaceuticalcomposition of claim 8, wherein the IRES has a sequence of an IRES fromTaura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus,Simian Virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus,Reticuloendotheliosis virus, Human poliovirus 1, Plautia stali intestinevirus, Kashmir bee virus, Human rhinovirus 2, Homalodisca coagulatavirus-1, Human Immunodeficiency Virus type 1, Homalodisca coagulatavirus-1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus,Hepatitis GB virus, Foot and mouth disease virus, Human enterovirus 71,Equine rhinitis virus, Ectropis obliqua picorna-like virus,Encephalomyocarditis virus, Drosophila C Virus, Human coxsackievirus B3,Crucifer tobamovirus, Cricket paralysis virus, Bovine viral diarrheavirus 1, Black Queen Cell Virus, Aphid lethal paralysis virus, Avianencephalomyelitis virus, Acute bee paralysis virus, Hibiscus chloroticringspot virus, Classical swine fever virus, Human FGF2, Human SFTPA1,Human AML1/RUNX1, Drosophila antennapedia, Human AQP4, Human AT1R, HumanBAG-1, Human BCL2, Human BiP, Human c-IAPl, Human c-myc, Human eIF4G,Mouse NDST4L, Human LEF1, Mouse HIF1 alpha, Human n.myc, Mouse Gtx,Human p27kipl, Human PDGF2/c-sis, Human p53, Human Pim-1, Mouse Rbm3,Drosophila reaper, Canine Scamper, Drosophila Ubx, Human UNR, MouseUtrA, Human VEGF-A, Human XIAP, Drosophila hairless, S. cerevisiaeTFIID, S. cerevisiae YAP1, tobacco etch virus, turnip crinkle virus,EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, Picobirnavirus, HCV QC64,Human Cosavirus E/D, Human Cosavirus F, Human Cosavirus JMY, RhinovirusNAT001, HRV14, HRV89, HRVC-02, HRV-A21, Salivirus A SH1, Salivirus FHB,Salivirus NG-J1, Human Parechovirus 1, Crohivirus B, Yc-3, RosavirusM-7, Shanbavirus A, Pasivirus A, Pasivirus A 2, Echovirus E14, HumanParechovirus 5, Aichi Virus, Hepatitis A Virus HA16, Phopivirus, CVA10,Enterovirus C, Enterovirus D, Enterovirus J, Human Pegivirus 2, GBV-CGT110, GBV-C K1737, GBV-C Iowa, Pegivirus A 1220, Pasivirus A 3,Sapelovirus, Rosavirus B, Bakunsa Virus, Tremovirus A, Swine Pasivirus1, PLV-CHN, Pasivirus A, Sicinivirus, Hepacivirus K, Hepacivirus A,BVDV1, Border Disease Virus, BVDV2, CSFV-PK15C, SF573 Dicistrovirus,Hubei Picorna-like Virus, CRPV, Apodemus Agrarius Picornavirus, CaprineKobuvirus, Parabovirus, Salivirus A BN5, Salivirus A BN2, Salivirus A02394, Salivirus A GUT, Salivirus A CH, Salivirus A SZ1, Salivirus FHB,CVB3, CVB1, Echovirus 7, CVB5, EVA71, CVA3, CVA12, EV24, or an aptamerto eIF4G.
 14. A pharmaceutical composition comprising: a. a circular RNApolynucleotide encoding a chimeric antigen receptor (CAR), and b. alipid nanoparticle (LNP) comprising an ionizable lipid represented byFormula (5A):

wherein: l is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6,7, 8, and 9; M₁ is a bond or M′; R₄ is unsubstituted C₁₋₃ alkyl, or—(CH₂)_(n)Q, in which Q is —OH, —NHC(S)N(R)₂, —NHC(O)N(R)₂, —N(R)C(O)R,—N(R)S(O)₂R, —N(R)R₈, —NHC(═NR₉)N(R)₂, —NHC(═CHR₉)N(R)₂, —OC(O)N(R)₂,—N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R, —N(OR)C(O)OR, —N(OR)C(O)N(R)₂,—N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂, —N(OR)C(═CHR₉)N(R)₂, heteroaryl orheterocycloalkyl; M and M′ are independently selected from —C(O)O—,—OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, —S—S—, an aryl group, and aheteroaryl group; and R₂ and R₃ are independently selected from thegroup consisting of H, C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl, wherein thecircular RNA polynucleotide is encapsulated in the LNP.
 15. Thepharmaceutical composition of claim 14, wherein the LNP comprises atleast one of the following: (a) polyethylene glycol (PEG)-lipid, (b)structural lipid, or (c) helper lipid.
 16. The pharmaceuticalcomposition of claim 15, wherein the PEG-lipid selected from the groupconsisting of DMG-PEG2000, PEG-c-DOMG, PEG-DMG, PEG-DSPE, DSPE-PEG2000,PEG-DSG, PEG-DAG, PEG-DPPE, PEG-c-DMA, PEG-DLPE, PEG-DMPE, PEG-DPPC, andPEG-DSPE.
 17. The pharmaceutical composition of claim 15, wherein theLNP comprises an ionizable lipid represented by Formula (5A), a helperlipid, a structural lipid, and a PEG-lipid, wherein the molar ratio ofionizable lipid:helper lipid:structural lipid:PEG-lipid is 16:1:4:1,62:4:33:1, or 50:10:38.5:1.5.
 18. The pharmaceutical composition ofclaim 14, wherein the ionizable lipid comprises:


19. The pharmaceutical composition of claim 14, wherein the circular RNApolynucleotide is made via circularization of a precursor RNApolynucleotide comprising, in the following order: a. a 5′ externalduplex forming region, b. a 3′ group I intron fragment, c. a 5′ internalspacer optionally comprising a 5′ internal duplex forming region, d. anIRES, e. an expression sequence encoding a CAR, f. a 3′ internal spaceroptionally comprising a 3′ internal duplex forming region, g. a 5′ groupI intron fragment, and h. a 3′ external duplex forming region.
 20. Thepharmaceutical composition of claim 14, wherein the circular RNApolynucleotide is made via circularization of a precursor RNApolynucleotide comprising, in the following order: a. a first polyAsequence, b. a 5′ external duplex forming region, c. a 5′ externalspacer, d. a 3′ group I intron fragment, e. a 5′ internal spacercomprising a 5′ internal duplex forming region, f. an IRES, g. anexpression sequence encoding a CAR, h. a 3′ internal spacer comprising a3′ internal duplex forming region, i. a 5′ group I intron fragment, j. a3′ external spacer, k. a 3′ external duplex forming region, and l. asecond polyA sequence.
 21. The pharmaceutical composition of claim 14,wherein the circular RNA polynucleotide is made via circularization of aprecursor RNA polynucleotide comprising, in the following order: a. afirst polyA sequence, b. a 5′ external spacer, c. a 3′ group I intronfragment, d. a 5′ internal spacer comprising a 5′ internal duplexforming region, e. an IRES, f. an expression sequence encoding a CAR, g.a 3′ internal spacer comprising a 3′ internal duplex forming region, h.a 5′ group I intron fragment, i. a 3′ external spacer, and j. a secondpolyA sequence.
 22. The pharmaceutical composition of claim 14, whereinthe circular RNA polynucleotide is made via circularization of aprecursor RNA polynucleotide comprising, in the following order: a. afirst polyA sequence, b. a 5′ external spacer, c. a 3′ group I intronfragment, d. a 5′ internal spacer comprising a 5′ internal duplexforming region, e. an IRES, f. an expression sequence encoding a CAR, g.a stop codon cassette, h. a 3′ internal spacer comprising a 3′ internalduplex forming region, i. a 5′ group I intron fragment, j. a 3′ externalspacer, and k. a second polyA sequence.
 23. The pharmaceuticalcomposition of claim 14, wherein the circular RNA polynucleotide is madevia circularization of a precursor RNA polynucleotide comprising, in thefollowing order: a. a binding site, b. a 5′ external spacer, c. a 3′group I intron fragment, d. a 5′ internal spacer comprising a 5′internal duplex forming region, e. an IRES, f. an expression sequenceencoding a CAR, g. a 3′ internal spacer comprising a 3′ internal duplexforming region, h. a 5′ group I intron fragment, i. a 3′ externalspacer, and j. a second binding site.